Integrated Transcriptomics and Metabolomics Analysis Promotes the Understanding of Adventitious Root Formation in Eucommia ulmoides Oliver.

As a primary approach to nutrient propagation for many woody plants, cutting roots is essential for the breeding and production of Eucommia ulmoides Oliver. In this study, hormone level, transcriptomics, and metabolomics analyses were performed on two E. ulmoides varieties with different adventitious root (AR) formation abilities. The higher JA level on the 0th day and the lower JA level on the 18th day promoted superior AR development. Several hub genes executed crucial roles in the crosstalk regulation of JA and other hormones, including F-box protein (EU012075), SAUR-like protein (EU0125382), LOB protein (EU0124232), AP2/ERF transcription factor (EU0128499), and CYP450 protein (EU0127354). Differentially expressed genes (DEGs) and metabolites of AR formation were enriched in phenylpropanoid biosynthesis, flavonoid biosynthesis, and isoflavonoid biosynthesis pathways. The up-regulated expression of PAL, CCR, CAD, DFR, and HIDH genes on the 18th day could contribute to AR formation. The 130 cis-acting lncRNAs had potential regulatory functions on hub genes in the module that significantly correlated with JA and DEGs in three metabolism pathways. These revealed key molecules, and vital pathways provided more comprehensive insight for the AR formation mechanism of E. ulmoides and other plants.

The chromosome-level genome of Eucommia ulmoides provides insights into sex differentiation and α-linolenic acid biosynthesis.

Eucommia ulmoides Oliver is a typical dioecious plant endemic to China that has great medicinal and economic value. Here, we report a high-quality chromosome-level female genome of E. ulmoides obtained by PacBio and Hi-C technologies. The size of the female genome assembly was 1.01 Gb with 17 pseudochromosomes and 31,665 protein coding genes. In addition, Hi-C technology was used to reassemble the male genome released in 2018. The reassembled male genome was 1.24 Gb with the superscaffold N50 (48.30 Mb), which was increased 25.69 times, and the number of predicted genes increased by 11,266. Genome evolution analysis indicated that E. ulmoides has undergone two whole-genome duplication events before the divergence of female and male, including core eudicot γ whole-genome triplication event (γ-WGT) and a recent whole genome duplication (WGD) at approximately 27.3 million years ago (Mya). Based on transcriptome analysis, EuAP3 and EuAG may be the key genes involved in regulating the sex differentiation of E. ulmoides. Pathway analysis showed that the high expression of ω-3 fatty acid desaturase coding gene EU0103017 was an important reason for the high α-linolenic acid content in E. ulmoides. The genome of female and male E. ulmoides presented here is a valuable resource for the molecular biological study of sex differentiation of E. ulmoides and also will provide assistance for the breeding of superior varieties.

Whole genome re-sequencing reveals the genetic diversity and evolutionary patterns of Eucommia ulmoides.

Eucommia ulmoides (E. ulmoides) is a deciduous perennial tree belonging to the order Garryales, and is known as "living fossil" plant, along with ginkgo (Ginkgo biloba), metaspaca (Metasequoia glyptostroboides) and dove tree (Davidia involucrata Baill). However, the genetic diversity and population structure of E. ulmoides are still  ambiguous nowdays. In this study, we re-sequenced the genomes of 12 E. ulmoides accessions from different major climatic geography regions in China to elucidate the genetic diversity, population structure and evolutionary pattern. By integration of phylogenetic analysis, principal component analysis and population structure analysis based on a number of high-quality SNPs, a total of 12 E. ulmoides accessions were clustered into four different groups. This result is consistent with their geographical location except for group samples from Shanghai and Hunan province. E. ulmoides accessions from Hunan province exhibited a closer genetic relationship with E. ulmoides accessions from Shanghai in China compared with other regions, which is also supported by the result of population structure analyses. Genetic diversity analysis further revealed that E. ulmoides samples in Shanghai and Hunan province were with higher genetic diversity than those in other regions in this study. In addition, we treated the E. ulmoides materials from Shanghai and Hunan province as group A, and the other materials from other places as group B, and then analyzed the evolutionary pattern of E. ulmoides. The result showed the significant differentiation (Fst = 0.1545) between group A and group B. Some candidate highly divergent genome regions were identified in group A by selective sweep analyses, and the function analysis of candidate genes in these regions showed that biological regulation processes could be correlated with the Eu-rubber biosynthesis. Notably, nine genes were identified from selective sweep regions. They were involved in the Eu-rubber biosynthesis and expressed in rubber containing tissues. The genetic diversity research and evolution model of E. ulmoides were preliminarily explored in this study, which laid the foundation for the protection of germplasm resources and the development and utilization of multipurpose germplasm resources in the future.

Analysis of chemotypes and their markers in leaves of core collections of Eucommia ulmoides using metabolomics.

The leaves of Eucommia ulmoides contain various active compunds and nutritional components, and have successively been included as raw materials in the Chinese Pharmacopoeia, the Health Food Raw Material Catalogue, and the Feed Raw Material Catalogue. Core collections of E. ulmoides had been constructed from the conserved germplasm resources basing on molecular markers and morphological traits, however, the metabolite diversity and variation in this core population were little understood. Metabolite profiles of E. ulmoides leaves of 193 core collections were comprehensively characterized by GC-MS and LC-MS/MS based non-targeted metabolomics in present study. Totally 1,100 metabolites were identified and that belonged to 18 categories, and contained 120 active ingredients for traditional Chinese medicine (TCM) and 85 disease-resistant metabolites. Four leaf chemotypes of the core collections were established by integrated uses of unsupervised self-organizing map (SOM), supervised orthogonal partial least squares discriminant analysis (OPLS-DA) and random forest (RF) statistical methods, 30, 23, 43, and 23 chemomarkers were screened corresponding to the four chemotypes, respectively. The morphological markers for the chemotypes were obtained by weighted gene co-expression network analysis (WGCNA) between the chenomarkers and the morphological traits, with leaf length (LL), chlorophyll reference value (CRV), leaf dentate height (LDH), and leaf thickness (LT) corresponding to chemotypes I, II, III, and IV, respectively. Contents of quercetin-3-O-pentosidine, isoquercitrin were closely correlated to LL, leaf area (LA), and leaf perimeter (LP), suggesting the quercetin derivatives might influence the growth and development of E. ulmoides leaf shape.

Genome-wide identification and expression pattern analysis of the ribonuclease T2 family in Eucommia ulmoides.

The 2',3'-cycling ribonuclease (RNase) genes are catalysts of RNA cleavage and include the RNase T2 gene family. RNase T2 genes perform important roles in plants and have been conserved in the genome of eukaryotic organisms. In this study we identified 21 EURNS genes in Eucommia ulmoides Oliver (E. ulmoides) and analyzed their structure, chromosomal location, phylogenetic tree, gene duplication, stress-related cis-elements, and expression patterns in different tissues. The length of 21 predicted EURNS proteins ranged from 143 to 374 amino acids (aa), their molecular weight (MW) ranged from 16.21 to 42.38 kDa, and their isoelectric point (PI) value ranged from 5.08 to 9.09. Two classifications (class I and class III) were obtained from the conserved domains analysis and phylogenetic tree. EURNS proteins contained a total of 15 motifs. Motif 1, motif 2, motif 3, and motif 7 were distributed in multiple sequences and were similar to the conserved domain of RNase T2. EURNS genes with similar structure and the predicted EURNS proteins with conserved motif compositions are in the same group in the phylogenetic tree. The results of RT-PCR and transcription data showed that EURNS genes have tissue-specific expression and exhibited obvious trends in different developmental stages. Gene duplication analysis results indicated that segment duplication may be the dominant duplication mode in this gene family. This study provides a theoretical basis for research on the RNase T2 gene family and lays a foundation for the further study of EURNS genes.

Chemotype classification and biomarker screening of male Eucommia ulmoides Oliv. flower core collections using UPLC-QTOF/MS-based non-targeted metabolomics.

BACKGROUND: In the Chinese health care industry, male Eucommia ulmoides Oliv. flowers are newly approved as a raw material of functional food. Core collections have been constructed from conserved germplasm resources based on phenotypic traits and molecular markers. However, little is known about these collections' phytochemical properties. This study explored the chemical composition of male E. ulmoides flowers, in order to provide guidance in the quality control, sustainable cultivation, and directional breeding of this tree species. METHODS: We assessed the male flowers from 22 core collections using ultra-performance liquid chromatography and quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) non-targeted metabolomics, and analyzed them using multivariate statistical methods including principal component analysis (PCA), hierarchical cluster analysis (HCA), and orthogonal partial least squares discriminant analysis (OPLS-DA). RESULTS: We annotated a total of 451 and 325 metabolites in ESI+ and ESI- modes, respectively, by aligning the mass fragments of the secondary mass spectra with those in the database. Four chemotypes were well established using the ESI+ metabolomics data. Of the 29 screened biomarkers, 21, 6, 19, and 5 markers corresponded to chemotypes I, II, III, and IV, respectively. More than half of the markers belonged to flavonoid and amino acid derivative classes. CONCLUSION: Non-targeted metabolomics is a suitable approach to the chemotype classification and biomarker screening of male E. ulmoides flower core collections. We first evaluated the metabolite profiles and compositional variations of male E. ulmoides flowers in representative core collections before establishing possible chemotypes and significant biomarkers denoting the variations. We used genetic variations to infer the metabolite compositional variations of male E. ulmoides flower core collections instead of using the geographical origins of the germplasm resources. The newly proposed biomarkers sufficiently classified the chemotypes to be applied for germplasm resource evaluation.

The Hardy Rubber Tree Genome Provides Insights into the Evolution of Polyisoprene Biosynthesis.

Eucommia ulmoides, also called hardy rubber tree, is an economically important tree; however, the lack of its genome sequence restricts the fundamental biological research and applied studies of this plant species. Here, we present a high-quality assembly of its ∼1.2-Gb genome (scaffold N50 = 1.88 Mb) with at least 26 723 predicted genes for E. ulmoides, the first sequenced genome of the order Garryales, which was obtained using an integrated strategy combining Illumina sequencing, PacBio sequencing, and BioNano mapping. As a sister taxon to lamiids and campanulids, E. ulmoides underwent an ancient genome triplication shared by core eudicots but no further whole-genome duplication in the last ∼125 million years. E. ulmoides exhibits high expression levels and/or gene number expansion for multiple genes involved in stress responses and the biosynthesis of secondary metabolites, which may account for its considerable environmental adaptability. In contrast to the rubber tree (Hevea brasiliensis), which produces cis-polyisoprene, E. ulmoides has evolved to synthesize long-chain trans-polyisoprene via farnesyl diphosphate synthases (FPSs). Moreover, FPS and rubber elongation factor/small rubber particle protein gene families were expanded independently from the H. brasiliensis lineage. These results provide new insights into the biology of E. ulmoides and the origin of polyisoprene biosynthesis.

[A genetic study of 15 STR loci in Chinese East Mongolian population].

OBJECTIVE: To investigate the genetic polymorphisms of 15 STRs loci in Chinese East Mongolian population. METHODS: DNA samples from 105 unrelated individuals in Chinese East Mongolian population were screened by Power Plex 16 System and ABI3100 Genetic Analyzer. RESULTS: The genotype frequencies of these 15 STR loci meet the Hardy-Weinberg expectation. The Matching probability of 15 STRs loci were between 0.0084-0.2169, discriminating power (DP) at 0.7831-0.9916, heterozygosity (H) at 0.5619-0.9231, power of exclusion (PE) at 0.4490-0.8444, polymorphism information content (PIC) at 0.5438-0.9178. The total DP of 15 STRs loci is 0.9999999999998. CONCLUSION: Our results showed that the 15 STRs loci of Power Plex 16 System are valuable STR loci genetic marker system having high DP and are useful in forensic case work in Chinese East Mongolian population.