The complex genome and adaptive evolution of polyploid Chinese pepper (Zanthoxylum armatum and Zanthoxylum bungeanum).

Zanthoxylum armatum and Zanthoxylum bungeanum, known as 'Chinese pepper', are distinguished by their extraordinary complex genomes, phenotypic innovation of adaptive evolution and species-special metabolites. Here, we report reference-grade genomes of Z. armatum and Z. bungeanum. Using high coverage sequence data and comprehensive assembly strategies, we derived 66 pseudochromosomes comprising 33 homologous phased groups of two subgenomes, including autotetraploid Z. armatum. The genomic rearrangements and two whole-genome duplications created large (~4.5 Gb) complex genomes with a high ratio of repetitive sequences (>82%) and high chromosome number (2n = 4x = 132). Further analysis of the high-quality genomes shed lights on the genomic basis of involutional reproduction, allomones biosynthesis and adaptive evolution in Chinese pepper, revealing a high consistent relationship between genomic evolution, environmental factors and phenotypic innovation. Our study provides genomic resources and new insights for investigating diversification and phenotypic innovation in Chinese pepper, with broader implications for the protection of plants under severe environmental changes.

Small RNA sequencing reveals various microRNAs involved in piperine biosynthesis in black pepper (Piper nigrum L.).

BACKGROUND: Black pepper (Piper nigrum L.), an important and long-cultivated spice crop, is native to South India and grown in the tropics. Piperine is the main pungent and bioactive alkaloid in the berries of black pepper, but the molecular mechanism for piperine biosynthesis has not been determined. MicroRNAs (miRNAs), which are classical endogenous noncoding small RNAs, play important roles in regulating secondary metabolism in many species, but less is known regarding black pepper or piperine biosynthesis. RESULTS: To dissect the functions of miRNAs in secondary metabolism especially in piperine biosynthesis, 110 known miRNAs, 18 novel miRNAs and 1007 individual targets were identified from different tissues of black pepper by small RNA sequencing. qRT-PCR and 5'-RLM-RACE experiments were conducted to validate the reliability of the sequencing data and predicted targets. We found 3 miRNAs along with their targets including miR166-4CL, miR396-PER and miR397-CCR modules that are involved in piperine biosynthesis. CONCLUSION: MiRNA regulation of secondary metabolism is a common phenomenon in plants. Our study revealed new miRNAs that regulate piperine biosynthesis, which are special alkaloids in the piper genus, and they might be useful for future piperine genetic improvement of black pepper.

Resequencing 93 accessions of coffee unveils independent and parallel selection during Coffea species divergence.

Coffea arabica, C. canephora and C. excelsa, with differentiated morphological traits and distinct agro-climatic conditions, compose the majority of the global coffee plantation. To comprehensively understand their genetic diversity and divergence for future genetic improvement requires high-density markers. Here, we sequenced 93 accessions encompassing these three Coffea species, uncovering 15,367,960 single-nucleotide polymorphisms (SNPs). These SNPs are unequally distributed across different genomic regions and gene families, with two disease-resistant gene families showing the highest SNP density, suggesting strong balancing selection. Meanwhile, the allotetraploid C. arabica exhibits greater nucleotide diversity, followed by C. canephora and C. excelsa. Population divergence (FST), population stratification and phylogeny all support strong divergence among species, with C. arabica and its parental species C. canephora being closer genetically. Scanning of genomic islands with elevated FST and structure-disruptive SNPs contributing to species divergence revealed that most of the selected genes in each lineage are independent, with a few being selected in parallel for two or three species, such as genes in root hair cell development, flavonols accumulation and disease-resistant genes. Moreover, some of the SNPs associated with coffee lipids exhibit significantly biased allele frequency among species, being valuable for interspecific breeding. Overall, our study not only uncovers the key population genomic patterns among species but also contributes a substantial genomic resource for coffee breeding.

De novo transcriptome sequencing of black pepper (Piper nigrum L.) and an analysis of genes involved in phenylpropanoid metabolism in response to Phytophthora capsici.

BACKGROUND: Piper nigrum L., or "black pepper", is an economically important spice crop in tropical regions. Black pepper production is markedly affected by foot rot disease caused by Phytophthora capsici, and genetic improvement of black pepper is essential for combating foot rot diseases. However, little is known about the mechanism of anti- P. capsici in black pepper. The molecular mechanisms underlying foot rot susceptibility were studied by comparing transcriptome analysis between resistant (Piper flaviflorum) and susceptible (Piper nigrum cv. Reyin-1) black pepper species. RESULTS: 116,432 unigenes were acquired from six libraries (three replicates of resistant and susceptible black pepper samples), which were integrated by applying BLAST similarity searches and noted by adopting Kyoto Encyclopaedia of Genes and Gene Ontology (GO) genome orthology identifiers. The reference transcriptome was mapped using two sets of digital gene expression data. Using GO enrichment analysis for the differentially expressed genes, the majority of the genes associated with the phenylpropanoid biosynthesis pathway were identified in P. flaviflorum. In addition, the expression of genes revealed that after susceptible and resistant species were inoculated with P. capsici, the majority of genes incorporated in the phenylpropanoid metabolism pathway were up-regulated in both species. Among various treatments and organs, all the genes were up-regulated to a relatively high degree in resistant species. Phenylalanine ammonia lyase and peroxidase enzyme activity increased in susceptible and resistant species after inoculation with P. capsici, and the resistant species increased faster. The resistant plants retain their vascular structure in lignin revealed by histochemical analysis. CONCLUSIONS: Our data provide critical information regarding target genes and a technological basis for future studies of black pepper genetic improvements, including transgenic breeding.

Chemical constituents from Piper hainanense and their cytotoxicities.

Two new compounds, (Z,R)-1-phenylethylcinnamate (1) and (1R,2R,3R,6S)-pipoxide (2) were isolated from the aerial part of Piper hainanense, along with 12 known compounds, including nine benzene derivatives (4-11), one isobutylamide (12), and two polyoxygenated cyclohexene derivatives (13-14). Their structures were elucidated on the basis of the HRESIMS, 1D and 2D NMR spectroscopic analyses, and ECD in cases of 2 and 3. The absolute configuration of ellipeiopsol B (3) was determined for the first time. All these compounds 1-14 were reported from the titled plant for the first time. Most of the isolates were tested for their cytotoxicities against five human cancer cell lines. Four of which, 2, 3, 9, 14 showed moderate bioactivities. Among them, the new compound 2 showed potential cytotoxicity against SMMC-7721, MCF-7, and SW-480 with IC50 values of 9.7, 15.0, and 13.2 µM, respectively.

Volatile organic compound emissions from different stages of Cananga odorata flower development.

Headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) was used to identify the volatile organic compounds (VOCs) of the different flower development stages of Cananga odorata for the evaluation of floral volatile polymorphism as a basis to determine the best time of harvest. Electronic nose results, coupled with discriminant factor analysis, suggested that emitted odors varied in different C. odorata flower development stages, including the bud, display-petal, initial-flowering, full-flowering, end-flowering, wilted-flower, and dried flower stages. The first two discriminant factors explained 97.52% of total system variance. Ninety-two compounds were detected over the flower life, and the mean Bray-Curtis similarity value was 52.45% among different flower development stages. A high level of volatile polymorphism was observed during flower development. The VOCs were largely grouped as hydrocarbons, esters, alcohols, aldehydes, phenols, acids, ketones, and ethers, and the main compound was ß-caryophyllene (15.05%-33.30%). Other identified compounds were ß-cubebene, D-germacrene, benzyl benzoate, and α-cubebene. Moreover, large numbers of VOCs were detected at intermediate times of flower development, and more hydrocarbons, esters, and alcohols were identified in the full-flowering stage. The full-flowering stage may be the most suitable period for C. odorata flower harvest.

Pipermotuoense, a new species of Piperaceae from Xizang, China.

Pipermotuoense X.W.Qin, F.Su & C.Y.Hao, a new species of Piperaceae from Xizang, China, is described and illustrated in this paper. The new species resembles P.yinkiangense and P.anisotis, but it can be readily distinguished from the compared species by several characteristics. Gonophyll leaves are chartaceous and the leaf secondary vein count is 7-9, with the outermost pair being very weak when there are nine veins. Additionally, the apical pair arises 2-4 cm above the base and the leaf base is asymmetrical, with bilateral petioles that cling and heal together. Pistillate floral bracts are sessile, with 3, 4 or 5 stigmas. The description of the new species includes photographs, detailed descriptions, notes on etymology, distribution and habitat, as well as comparisons with morphologically similar species.

Staurantherafloribunda, a new species of Gesneriaceae from Yunnan, China.

Staurantherafloribunda F.Su, C.Y.Hao & K.Tan, a new species of Gesneriaceae from Yunnan, China, is described and illustrated here. It is morphologically similar to S.grandifolia Benth. in the shape of corolla, stigma, leaves and the number of stamens. However, it can be readily distinguished from the compared species by its dense cymes, leaf indumentum, lack of a corolla spur, calyx colour and stamen shape. The description of the new species, photographs, detailed descriptions, notes on etymology, distribution and habitat, as well as comparisons with morphologically similar species, are provided.

Effects of Cabya (Piper retrofractum Vahl.) Fruit Developmental Stage on VOCs.

The differences in VOCs can affect the flavor and medicinal value of cabya, and the flavor changes that occur in stages as the fruit develops are currently unknown. In order to investigate the influence of the developmental stage on the aroma composition of cabya essential oil, VOCs at each of the four developmental stages were analyzed by steam distillation (SD) extraction combined with GC-MS detection. The similarities and differences in fruit composition among the developmental stages were evaluated using hierarchical cluster analysis (HCA) and principal component analysis (PCA). A total of 60 VOCs, mainly alcohols, alkenes and alkanes, were identified across all of the developmental stages. The most acidic substances were detected in phase A and have a high medicinal value. There was no significant difference between the B and C phases, and the alcohols in those phases mainly promoted terpenoid synthesis in the D phase. Constituents during the D phase were mainly alkenes, at 57.14%, which contributed significantly to the aroma of the essential oil. PCA and HCA both were able to effectively differentiate the cabya fruit developmental stages based on the SD-GC-MS data. In summary, this study investigated the flavor variation characteristics and the diversity of VOCs in cabya fruits at different developmental stages, and its findings can provide a reference for developing essential oil products for different uses and determining appropriate stages for harvesting cabya resources.

Comparative analysis of the chloroplast genomes of eight Piper species and insights into the utilization of structural variation in phylogenetic analysis.

With more than 2000 species, Piper is regarded as having high medicinal, cosmetic, and edible value. There also remain some taxonomic and evolutionary uncertainties about the genus. This study performed chloroplast genome sequencing of eight poorly studied Piper species and a comparative analysis with black pepper (Piper nigrum). All examined species were highly similar in gene content, with 79 protein-coding genes, 24 tRNAs, and four rRNAs. They also harbored significant structural differences: The number of SSRs ranged from 63 to 87, over 10,000 SNPs were detected, and over 1,000 indels were found. The spatial distribution of structural differences was uneven, with the IR and LSC being relatively more conserved and the SSC region highly variable. Such structural variations of the chloroplast genome can help in evaluating the phylogenetic relationships between species, deciding some hard-to-distinguish evolutionary relationships, or eliminating improper markers. The SSC region may be evolving at high speed, and some species showed a high degree of sequence variation in the SSC region, which seriously affected marker sequence detection. Conversely, CDS sequences tended to lack variation, and some CDSs can serve as ideal markers for phylogenetic reconstruction. All told, this study provides an effective strategy for selecting chloroplast markers, analyzing difficult-to-distinguish phylogenetic relationships and avoiding the taxonomic errors caused by high degree of sequence variations.

An Effective Inoculation Method for Phytophthora capsici on Black Pepper Plants.

Piper nigrum L. (black pepper) is a typical woody vine that is an economically important spice crop across the world. Black pepper production is significantly impacted by root rot disease caused by Phytophthora capsici, which has seriously influenced the industry development as a "choke point" problem. However, the molecular genetic mechanism of resistance in black pepper is unclear, leading to slow progress in the development of new black pepper varieties. An effective inoculation and precise sampling system for Phytophthora capsici on black pepper plants is essential for studying this plant-pathogen interaction. The main aim of this study is to demonstrate a detailed methodology where the basal head of black pepper is inoculated with Phytophthora capsici, while also providing a reference for the inoculation of woody vine plants. The basal head of the black pepper plant was pinpricked to damage it, and mycelial pellets covered the three holes to retain the moisture so the pathogen could infect the plant well. This method provides a better way of solving the instability that is caused by traditional inoculation methods including soil drench or root dipping. It also provides a promising means for studying the mode of action between plants and other soil-borne plant pathogens in agricultural precision breeding.

Comparative Transcriptome and Metabolome Analysis of Resistant and Susceptible Piper Species Upon Infection by the Oomycete Phytophthora Capsici.

Phytophthora capsici is a destructive oomycete pathogen that causes devastating disease in black pepper, resulting in a significant decline in yield and economic losses. Piper nigrum (black pepper) is documented as susceptible to P. capsici, whereas its close relative Piper flaviflorum is known to be resistant. However, the molecular mechanism underlying the resistance of P. flaviflorum remains obscure. In this study, we conducted a comparative transcriptome and metabolome analysis between P. flaviflorum and P. nigrum upon P. capsici infection and found substantial differences in their gene expression profiles, with altered genes being significantly enriched in terms relating to plant-pathogen interaction, phytohormone signal transduction, and secondary metabolic pathways, including phenylpropanoid biosynthesis. Further metabolome analysis revealed the resistant P. flaviflorum to have a high background endogenous ABA reservoir and time-course-dependent accumulation of ABA and SA upon P. capsici inoculation, while the susceptible P. nigrum had a high background endogenous IAA reservoir and time-course-dependent accumulation of JA-Ile, the active form of JA. Investigation of the phenylpropanoid biosynthesis metabolome further indicated the resistant P. flaviflorum to have more accumulation of lignin precursors than the susceptible P. nigrum, resulting in a higher accumulation after inoculation. This study provides an overall characterization of biologically important pathways underlying the resistance of P. flaviflorum, which theoretically explains the advantage of using this species as rootstock for the management of oomycete pathogen in black pepper production.

Genome-Wide Identification and Functional Exploration of SBP-Box Gene Family in Black Pepper (Piper nigrum L.).

Black pepper (Piper nigrum L.), is dubbed "the King of Spices". However, the lack of genic knowledge has limited the understanding of its physiological processes and hindered the development of its molecular breeding. The SBP-box gene family is an important family in plant development and integrates multiple physiological processes. Here, we made a genome-wide identification of the pepper SBP-box gene family to provide evolutionary and functional information about this conserved transcription factor. In total, 34 SBP genes were identified in pepper. All these pepper SBP genes were clustered into eight groups, and one pepper group was not found in Arabidopsis thaliana. Segment duplications played the most important role in the expansion process of pepper SBP genes, and all these duplications were subjected to purifying selection. Half of pepper SBP genes were found miR156 target sites, and 17 miR156s were predicted. The tissue expression analysis revealed the differential expression of pepper SBP genes. Eleven SBP genes were found in four co-expression networks, and the GO enrichment further provides a functional prediction for pepper SBP genes. This study lays a foundation for further studies of pepper and provides a valuable reference for functional mining of pepper SBP genes.

Transcriptional profiling reveals differentially expressed genes involved in lipid biosynthesis during cacao seed development.

Theobroma cacao is a plant of economic value due to the use of its seed lipid for chocolate, confectionery, and cosmetic industries. The seed lipid contains a stable ratio of saturated and unsaturated fatty acids, which determines its unique melting temperature. However, little is known about the molecular mechanism determining the fatty acid ratio and lipid content in cacao. To gain insight into the unique properties of lipid synthesis in cacao, biochemical and transcriptomic approaches were used to compare the lipid accumulation between high and low lipid content cacao accessions. Lipid accumulation rates and lipid content were different between the two accessions. Moreover, differentially expressed genes were detected between high and low lipid content cacao accessions. The data allowed the identification of distinct candidate genes and furthered our understanding of lipid accumulation, potentially explaining the differences in lipid content between various cacao accessions. The results might be used to develop molecular tools and engineer alternative pathways for cacao breeding with improved lipid production potentials.

The chromosome-scale reference genome of black pepper provides insight into piperine biosynthesis.

Black pepper (Piper nigrum), dubbed the 'King of Spices' and 'Black Gold', is one of the most widely used spices. Here, we present its reference genome assembly by integrating PacBio, 10x Chromium, BioNano DLS optical mapping, and Hi-C mapping technologies. The 761.2 Mb sequences (45 scaffolds with an N50 of 29.8 Mb) are assembled into 26 pseudochromosomes. A phylogenomic analysis of representative plant genomes places magnoliids as sister to the monocots-eudicots clade and indicates that black pepper has diverged from the shared Laurales-Magnoliales lineage approximately 180 million years ago. Comparative genomic analyses reveal specific gene expansions in the glycosyltransferase, cytochrome P450, shikimate hydroxycinnamoyl transferase, lysine decarboxylase, and acyltransferase gene families. Comparative transcriptomic analyses disclose berry-specific upregulated expression in representative genes in each of these gene families. These data provide an evolutionary perspective and shed light on the metabolic processes relevant to the molecular basis of species-specific piperine biosynthesis.

[The applications of fourier transform infrared spectroscopy in resolving some taxonomic doubts of Caprifoliaceae].

Fourier transform infrared spectroscopy (FTIR) provides biochemical profiles containing overlapping signals from a majority of the compounds that are present when whole.samples are analyzed. The spectra of the xylem of 112 samples belonging to 12 species and 5 genera in Caprifoliaceae were determined directly with FTIR spectrometry and OMNI-sampler. Based on the indices of wave number -absorbance, the phylogenetic relationships of the 12 species were analyzed by the methods of principal coordinate analysis (PCO) and cluster analysis. The results showed that the infrared spectra of the 12 plant species are finger-print-like patterns which are highly typical for different taxa. Genus Heptacodium has close relation with the tribe Linnaeeae, and Lonicereae, showing that its systematic position is probably located between the two tribes. There is no obvious difference between Viburnum and Sambucus, so the authors thought it would be better to put the two genera in Caprifoliaceae. Therefore, the infrared spectra are of taxonomic value at the level of species and genera, and this technique could be widely used for identification and classification of other taxa when standard spectra are available.

Chemical Composition and Acetylcholinesterase Inhibitory Activity of Essential Oils from Piper Species.

The essential oils (EOs) derived from aromatic plants such as Piper species are considered to play a role in alleviating neuronal ailments that are associated with inhibition of acetylcholinesterase (AChE). The chemical compositions of 23 EOs prepared from 16 Piper spp. were analyzed by both gas chromatography with a flame ionization detector (GC-FID) and gas chromatography-mass spectrometry (GC-MS). A total of 76 compounds were identified in the EOs from the leaves and stems of 19 samples, while 30 compounds were detected in the EOs from the fruits of four samples. Sesquiterpenes and phenylpropanoids were found to be rich in these EOs, of which asaricin, caryophyllene, caryophyllene oxide, isospathulenol, (+)-spathulenol, and ß-bisabolene are the major constituents. The EOs from the leaves and stems of Piper austrosinense, P. puberulum, P. flaviflorum, P. betle, and P. hispidimervium showed strong AChE inhibitory activity with IC50 values in the range of 1.51 to 13.9 mg/mL. A thin-layer chromatography (TLC) bioautography assay was employed to identify active compound(s) in the most active EO from P. hispidimervium. The active compound was isolated and identified as asaricin, which gave an IC50 value of 0.44 ± 0.02 mg/mL against AChE, comparable to galantamine with an IC50 0.15 ± 0.01 mg/mL.

Transcriptome and selected metabolite analyses reveal points of sugar metabolism in jackfruit (Artocarpus heterophyllus Lam.).

Artocarpus heterophyllus Lam., commonly known as jackfruit, produces the largest tree-borne fruit known thus far. The edible part of the fruit develops from the perianths, and contains many sugar-derived compounds. However, its sugar metabolism is poorly understood. A fruit perianth transcriptome was sequenced on an Illumina HiSeq 2500 platform, producing 32,459 unigenes with an average length of 1345nt. Sugar metabolism was characterized by comparing expression patterns of genes related to sugar metabolism and evaluating correlations with enzyme activity and sugar accumulation during fruit perianth development. During early development, high expression levels of acid invertases and corresponding enzyme activities were responsible for the rapid utilization of imported sucrose for fruit growth. The differential expression of starch metabolism-related genes and corresponding enzyme activities were responsible for starch accumulated before fruit ripening but decreased during ripening. Sucrose accumulated during ripening, when the expression levels of genes for sucrose synthesis were elevated and high enzyme activity was observed. The comprehensive transcriptome analysis presents fundamental information on sugar metabolism and will be a useful reference for further research on fruit perianth development in jackfruit.

[Identification of soybean varieties by direct determination of FTIR spectrum].

The spectra of cotyledons and outer layer parts of skins of eight kinds of soybeans were determined by FTIR spectrometry with OMNI-sampler directly, fast and accurately. The absorption peaks of the spectra w ere analyzed. Results from the comparison of the spectra showed some differences in the intensity and the shape of absorption peaks of their FTIR spectra, especially in the range of 1800-1200 cm(-1), which mainly reflected the substituent nature of amide I and II of protein. The major differences of spectra in this region might result from the differences in genes. So FTIR spectra could be used for the identification of soybean varieties.

Gene structure, phylogeny and expression profile of the sucrose synthase gene family in cacao (Theobroma cacao L.).

In higher plants, sucrose synthase (Sus, EC 2.4.1.13) is widely considered as a key enzyme involved in sucrose metabolism. Although, several paralogous genes encoding different isozymes of Sus have been identified and characterized in multiple plant genomes, to date detailed information about the Sus genes is lacking for cacao. This study reports the identification of six novel Sus genes from economically important cacao tree. Analyses of the gene structure and phylogeny of the Sus genes demonstrated evolutionary conservation in the Sus family across cacao and other plant species. The expression of cacao Sus genes was investigated via real-time PCR in various tissues, different developmental phases of leaf, flower bud and pod. The Sus genes exhibited distinct but partially redundant expression profiles in cacao, with TcSus1, TcSus5 and TcSus6, being the predominant genes in the bark with phloem, TcSus2 predominantly expressing in the seed during the stereotype stage. TcSus3 and TcSus4 were significantly detected more in the pod husk and seed coat along the pod development, and showed development dependent expression profiles in the cacao pod. These results provide new insights into the evolution, and basic information that will assist in elucidating the functions of cacao Sus gene family.