Leaf phenotypic variation and its response to environmental factors in natural populations of Eucommia ulmoides.

BACKGROUND: Eucommia ulmoides leaves have high medicinal and economic value as a dual-purpose substance for medicine and food. Employing leaves from 13 natural populations of Eucommia ulmoides as research objects, this study reveals the variation patterns of intra-specific and inter-specific trait variation and explores the response of leaf characteristics to geographical and climatic changes, aiming to provide a scientific basis for the efficient utilization of leaf resources and the breeding of superior varieties. RESULTS: Descriptive statistical analysis and nested analysis of variance showed significant differences in 11 leaf traits of Eucommia ulmoides inter-populations and intra-populations, with an average coefficient of variation of 17.45%. The coefficient of variation for average leaf phenotypic traits is 20.77%, and the leaf phenotypic variation is mainly from the variation intra-populations. Principal component analysis reveals that the cumulative contribution rate of the top three principal components which mainly contributed to the phenotypic variation of Eucommia ulmoides leaves reached 74.98%, which could be sorted into size traits (34.57%), color traits (25.82%) and shape traits (14.58%). In addition, correlation analysis expresses there is a specific co-variation pattern among leaf traits, with a strong connection between shape, size, and color traits. Geographic and climatic distances are significantly correlated, and mantel test and correlation analysis indicate that leaf traits of Eucommia ulmoides are mainly influenced by altitude. With the increase of altitude, the leaves become smaller. Partial correlation analysis shows that after controlling climate factors, the correlation between some characters and geographical factors disappears significantly. Temperature and precipitation have a great influence on the variation of leaf phenotypic traits, and the larger the leaves are in areas with high temperature and heavy rainfall. CONCLUSIONS: These findings contribute to a further understanding of the leaf morphological characteristics of Eucommia ulmoides and the extent to which the environment influences leaf trait variation. They can provide a scientific basis for the protection and application of Eucommia ulmoides leaf resources in the future.

Supplementary UV-B Radiation Effects on Photosynthetic Characteristics and Important Secondary Metabolites in Eucommia ulmoides Leaves.

To explore the effects of ultraviolet light supplementation on the photosynthetic characteristics and content of secondary metabolites in the leaves of Eucommia ulmoides Oliver (E. ulmoides), the effects of supplementary UV-B (sUV-B) radiation on the medicinally active components of E. ulmoides were comprehensively evaluated. In our study, we selected leaves of five-year-old E. ulmoides seedlings as experimental materials and studied the effect of supplemental ultraviolet-B (sUV-B) radiation on growth, photosynthetic parameters, photosynthetic pigments, fluorescence parameters, and secondary metabolites of E. ulmoides using multivariate analysis. The results showed that the leaf area and the number of branches increased after sUV-B radiation, which indicated that sUV-B radiation was beneficial to the growth of E. ulmoides. The contents of chlorophyll a and chlorophyll b increased by 2.25% and 4.25%, respectively; the net photosynthetic rate increased by 5.17%; the transpiration rate decreased by 35.32%; the actual photosynthetic efficiency increased by 10.64%; the content of the secondary metabolite genipin increased by 12.9%; and the content of chlorogenic acid increased by 75.03%. To identify the genes that may be related to the effects of sUV-B radiation on the growth and development of E. ulmoides leaves and important secondary metabolites, six cDNA libraries were prepared from natural sunlight radiation and sUV-B radiation in E. ulmoides leaves. Comparative analysis of both transcriptome databases revealed a total of 3698 differential expression genes (DEGs), including 1826 up-regulated and 1872 down-regulated genes. According to the KOG database, the up-regulated unigenes were mainly involved in signal transduction mechanisms [T] and cell wall/membrane biogenesis [M]. It is also involved in plant hormone signal transduction and phenylpropanoid biosynthesis metabolic pathways by the KEGG pathway, which might further affect the physiological indices and the content of chlorogenic acid, a secondary metabolite of E. ulmoides. Furthermore, 10 candidate unigenes were randomly selected to examine gene expression using qRT-PCR, and the six libraries exhibited differential expression and were identical to those obtained by sequencing. Thus, the data in this study were helpful in clarifying the reasons for leaf growth after sUV-B radiation. And it was beneficial to improve the active components and utilization rate of E. ulmoides after sUV-B radiation.

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.

Nocardiopsis eucommiae sp. nov., a novel endophytic actinomycete isolated from leaves of Eucommia ulmoides Oliv.

A novel Gram-stain-positive, endophytic actinobacterium, designated strain HDS5T, was isolated from leaves of Eucommia ulmoides Oliv. collected from Changde City, Hunan Province, PR China. Strain HDS5T produced yellowish oil green substrate mycelia on Gause's synthetic medium, which also carried yellowish oil green aerial hyphae, fragmenting into rod-shaped elements with smooth surfaces. Strain HDS5T grew at pH 5.0-11.0 (optimum, pH 7), at 20-40 °C (optimum, 28 °C) and in the presence of 0-8.0% NaCl (w/v; optimum, 0-1.0 %). Whole-cell hydrolysates contained meso-diaminopimelic acid as the diagnostic amino acid and no diagnostic sugars. The predominant fatty acids were iso-C16:0 and C18 : 1 ω9c. The menaquinones were MK-10(H2), MK-10(H4) and MK-10(H6). Strain HDS5T showed high 16S rRNA gene sequence similarity to Nocardiopsis prasina DSM 43845T (99.72 %), Nocardiopsis ganjiahuensis DSM 45031T (99.31 %), Nocardiopsis exhalans JCM 11759T (99.17 %), Nocardiopsis alba DSM 43377T (99.11 %), Nocardiopsis metallicus KBS6T (99.11 %), Nocardiopsis valliformis DSM 45023T (99.04 %), Nocardiopsis listeri NBRC 13360T (98.97 %), Nocardiopsis lucentensis DSM 44048T (98.83 %), Nocardiopsis terrae YIM 90022T (98.83 %) and <98.7 % similarities to other type strains. Phylogenetic analysis of 16S rRNA gene sequences and whole-genome sequences showed that strain HDS5T was closely related to N. prasina DSM 43845T. However, the average nucleotide identity based on blast and digital DNA-DNA hybridization values between them were determined to be 90.1 and 40.9 %, respectively, below the threshold of 95-96 and 70 % for the delineation of prokaryotic genomic species, suggesting that strain HDS5T represents a novel Nocardiopsis species. Furthermore, the morphological and physio-biochemical characteristics were sufficient to distinguish strain HDS5T from N. prasina DSM 43845T. Consequently, based on phenotypic and genotypic characteristics, strain HDS5T represents a new Nocardiopsis species, for which the name Nocardiopsis eucommiae sp. nov. is proposed. The type strain is HDS5T (=MCCC 1K06172T=JCM 34707T).

Identification, evolution and expression analysis of WRKY gene family in Eucommia ulmoides.

The WRKY transcription factors is one of the largest families of transcription factors (TFs) in plants and involved in multiple biological processes. However, the role of the WRKY family had not been reported in Eucommia ulmoides. In this study, 45 WRKY genes (EuWRKY1-45) with conserved WRKY domain were identified in E. ulmoides and classified into three groups. The group II was further divided into five subgroups based on phylogenetic analysis, and each clade was well supported by the conserved motifs. All the genes were located on 34 different scaffolds respectively. A number of development-, light-, hormone-, and stress-related elements were randomly distributed in the promoter sequences of EuWRKYs. Expression profiles indicated that EuWRKY genes were involved in leaf development, and majority of EuWRKYs genes were highly expressed in leaf buds. Co-expression analysis of WRKYs suggested an intricate interplay of growth-related responses. EuWRKY4 was involved in a complex proteins interaction network. Collectively, our results provide extensive insights into the WRKY gene family, thereby contributing to the screening of additional candidate genes in E. ulmoides.

Overexpression of the Eucommia~ulmoides Aquaporin, EuPIP1;1, Promotes Leaf Growth, Flowering and Bolting, and Stress Tolerance in Arabidopsis.

Plasma membrane intrinsic protein (PIP) is one of the largest subfamilies of Aquaporins (AQPs) and plays an important role in plant growth and development, and resistance to abiotic stress. In this study, the full length of the EuPIP1;1 cDNA was cloned from Eucommia ulmoides using the rapid amplification of cDNA ends (RACE) method. The EuPIP1;1 gene was induced by drought treatment and expressed in all tested tissues, with the highest expression level in fruit. The subcellular localization showed that EuPIP1;1 was located in the plasma membrane. Constitutive overexpression of EuPIP1;1 in Arabidopsisthaliana could promote leaf growth and development, and accelerate bolting and flowering. Six genes related to growth and flowering (AtPIF4, AtTCP14, AtCRY1, AtCRY2, AtFCA and AtFT) were significantly up-regulated in transgenic lines. Further, EuPIP1;1 gene improved resistance to drought and salt stress in transgenic Arabidopsis. Under drought and salt stress treatment, the transgenic lines had a higher germination rate and accumulation of osmotic substances, lower membrane damage, and could maintain ion homeostasis. Our results suggest that EuPIP1;1 plays an essential role in plant growth and development and in the response to drought and salt stress.

[Identification of key enzyme genes involved in biosynthesis pathways of lignan and lignin in Eucommia ulmoides based on transcriptome assembly].

Lignan is the main medicinal component of Eucommia ulmoides, and lignin is involved in the defense of plants against diseases and insect pests.They are synthesized from coniferyl alcohol with the help of dirigent(DIR) and peroxidase(POD), respectively.In this study, transcriptome assembly of stems and leaves of E.ulmoides was performed, yielding 112 578 unigenes.Among them, 70 459 were annotated in seven databases.A total of 59 unigenes encodes 11 key enzymes in the biosynthesis pathways of lignin and lignin, of which 11 encode POD and 8 encode DIR.A total of 13 unigenes encoding transcription factors are involved in phenylpropanoid metabolism. Compared with leaves of E.ulmoides, 7 575 unigenes were more highly expressed in stems, of which 462 were involved in phenylpropanoid biosynthesis.Our results extend the public transcriptome dataset of E.ulmoides, which provide valuable information for the analysis of biosynthesis pathways of lignan and lignin in E.ulmoides and lay a foundation for further study on the functions and regulation mechanism of key enzymes in lignan and lignin biosynthesis pathways.

Genome-wide association study reveals the genes associated with the leaf inclusion contents in Chinese medical tree Eucommia ulmoides.

Eucommia ulmoides is an economic tree that can biosynthesize secondary metabolites with pharmacological functions. Genetic basis of biosynthesis of these compounds is almost unknown. Therefore, genomic-wide association study was performed to exploit the genetic loci maybe involved in biosynthetic pathways of 5 leaf inclusions (aucubin, chlorogenic acid, gutta-percha, polyphenols, total flavonoids). It was shown that contents of the 5 leaf metabolites have a wide variation following normal distribution. A total of 2 013 102 single nucleotide polymorphism (SNP) markers were identified in a population containing 62 individual clones. Through genome-wide association study analysis, many SNP loci were identified perhaps associated with phenotypes of the leaf inclusions. Higher transcriptional levels of the candidate genes denoted by significant SNPs in leaves suggested they may be involved in biosynthesis of the leaf inclusions. These genetic loci provide with invaluable information for further studies on the gene functions in biosynthesis of the leaf inclusions and selective breeding of the plus trees.

Transcriptome analysis of terpenoid biosynthetic genes and simple sequence repeat marker screening in Eucommia ulmoides.

Trans-polyisoprene rubber is produced in the tissues of leaves, bark, and fruit of Eucommia ulmoides and is considered an important energy source. Transcript profiles of two tissues from E. ulmoides cv. Qinzhong No. 3, leaf and fruit, were analysed using the Illumina HiSeq 2000 system. In total, 104 million clean reads were obtained and assembled into 58,863 unigenes. Through gene functional classification, 28,091 unigenes (47.72%) were annotated and 65 unigenes have been hypothesized to encode proteins involved in terpenoid biosynthesis. In addition, 10,041 unigenes were detected as differentially expressed unigenes, and 29 of them were putatively related to terpenoid biosynthesis. The synthesis of trans-polyisoprene rubbers in E. ulmoides was hypothesised to be dominated by the mevalonate pathway. Farnesyl diphosphate synthase 2 (FPPS2) was considered a key component in the biosynthesis of trans-polyprenyl diphosphate. Rubber elongation factor 3 (REF3) might be involved in stabilising the membrane of rubber particles in E. ulmoides. To date, 351 simple sequence repeats (SSRs) were validated as polymorphisms from eight E. ulmoides plants (two parent plants and six F1 individuals), and these could act as molecular markers for genetic map density increase and breeding improvement of E. ulmoides.

Dynamic Changes in Metabolite Accumulation and the Transcriptome during Leaf Growth and Development in Eucommia ulmoides.

Eucommia ulmoides Oliver is widely distributed in China. This species has been used mainly in medicine due to the high concentration of chlorogenic acid (CGA), flavonoids, lignans, and other compounds in the leaves and barks. However, the categories of metabolites, dynamic changes in metabolite accumulation and overall molecular mechanisms involved in metabolite biosynthesis during E. ulmoides leaf growth and development remain unknown. Here, a total of 515 analytes, including 127 flavonoids, 46 organic acids, 44 amino acid derivatives, 9 phenolamides, and 16 vitamins, were identified from four E. ulmoides samples using ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) (for widely targeted metabolites). The accumulation of most flavonoids peaked in growing leaves, followed by old leaves. UPLC-MS analysis indicated that CGA accumulation increased steadily to a high concentration during leaf growth and development, and rutin showed a high accumulation level in leaf buds and growing leaves. Based on single-molecule long-read sequencing technology, 69,020 transcripts and 2880 novel loci were identified in E. ulmoides. Expression analysis indicated that isoforms in the flavonoid biosynthetic pathway and flavonoid metabolic pathway were highly expressed in growing leaves and old leaves. Co-expression network analysis suggested a potential direct link between the flavonoid and phenylpropanoid biosynthetic pathways via the regulation of transcription factors, including MYB (v-myb avian myeloblastosis viral oncogene homolog) and bHLH (basic/helix-loop-helix). Our study predicts dynamic metabolic models during leaf growth and development and will support further molecular biological studies of metabolite biosynthesis in E. ulmoides. In addition, our results significantly improve the annotation of the E. ulmoides genome.

Whole-Genome Comparison Reveals Heterogeneous Divergence and Mutation Hotspots in Chloroplast Genome of Eucommia ulmoides Oliver.

Eucommia ulmoides (E. ulmoides), the sole species of Eucommiaceae with high importance of medicinal and industrial values, is a Tertiary relic plant that is endemic to China. However, the population genetics study of E. ulmoides lags far behind largely due to the scarcity of genomic data. In this study, one complete chloroplast (cp) genome of E. ulmoides was generated via the genome skimming approach and compared to another available E. ulmoides cp genome comprehensively at the genome scale. We found that the structure of the cp genome in E. ulmoides was highly consistent with genome size variation which might result from DNA repeat variations in the two E. ulmoides cp genomes. Heterogeneous sequence divergence patterns were revealed in different regions of the E. ulmoides cp genomes, with most (59 out of 75) of the detected SNPs (single nucleotide polymorphisms) located in the gene regions, whereas most (50 out of 80) of the indels (insertions/deletions) were distributed in the intergenic spacers. In addition, we also found that all the 40 putative coding-region-located SNPs were synonymous mutations. A total of 71 polymorphic cpDNA fragments were further identified, among which 20 loci were selected as potential molecular markers for subsequent population genetics studies of E. ulmoides. Moreover, eight polymorphic cpSSR loci were also developed. The sister relationship between E. ulmoides and Aucuba japonica in Garryales was also confirmed based on the cp phylogenomic analyses. Overall, this study will shed new light on the conservation genomics of this endangered plant in the future.

Genome-wide screening of long non-coding RNAs involved in rubber biosynthesis in Eucommia ulmoides.

Increasing evidence indicates that long non-coding RNAs (lncRNAs) play pivotal roles in regulatory networks controlling plant and animal gene expression. However, lncRNA roles in regulating rubber biosynthesis in Eucommia ulmoides, an emerging source of natural rubber (Eu-rubber), are currently unknown. Here, we report on RNA deep-sequencing of E. ulmoides fruits at two developmental stages. Based on application of a stringent pipeline, 29,103 lncRNAs and 9,048 transcripts of uncertain coding potential (TUCPs) were identified. Two differentially expressed (DE) TUCPs appear to simultaneously regulate 12 protein-coding genes involved in Eu-rubber biosynthesis (GIEBs), as well as 95 DE genes. Functional categorization of these 95 DE genes indicated their involvement in subcellular microstructures and cellular processes, such as cell wall, cell division, and growth. These DE genes may participate in the differentiation and development of laticifers, where Eu-rubber is synthesized. A model is proposed in which "commanders" (DE TUCPs) direct the "builders" (DE genes) to construct a "storehouse" of materials needed for Eu-rubber synthesis, and the "workers" (GIEBs) to synthesize Eu-rubber. These findings provide insights into both cis- and trans-polyisoprene biosynthesis in plants, laying the foundation for additional studies of this crucial process.

Identification of the Sex-Biased Gene Expression and Putative Sex-Associated Genes in Eucommia ulmoides Oliver Using Comparative Transcriptome Analyses.

Eucommia ulmoides is a model representative of the dioecious plants with sex differentiation at initiation. Nevertheless, the genetic mechanisms of sexual dimorphism and sex determination in E. ulmoides remain poorly understood. In this study de novo transcriptome sequencing on Illumina platform generated >45 billion high-quality bases from fresh leaves of six male and female individuals of E. ulmoides. A total of 148,595 unigenes with an average length of 801 base-pairs (bp) were assembled. Through comparative transcriptome analyses, 116 differentially expressed genes (DEGs) between the males and the females were detected, including 73 male-biased genes and 43 female-biased genes. Of these DEGs, three female-biased genes were annotated to be related with the sexually dimorphic gutta content in E. ulmoides. One male-biased DEG was identified as putative MADS box gene APETALA3, a B class floral organ identity gene in the flowering plants. SNPs calling analyses further confirmed that the APETALA3-like gene was probably involved in the sex determination in E. ulmoides. Four other male-biased DEGs were potential sex-associated genes as well with segregated SNPs in accord with sex type. In addition, the SNPs density was 1.02 per kilobase (kb) in the expressed genes of E. ulmoides, implying a relatively high genetic diversity.

Isolation and characterization of microsatellite markers for Eucommia ulmoides (Eucommiaceae), an endangered tree, using next-generation sequencing.

Eucommia ulmoides Oliver, a single extant species of Eucommiaceae, is an endemic dioecious tree in China. The natural resources of E. ulmoides have rapidly declined in recent years because of the over-collection of its cortex. To design a suitable protection strategy, it is necessary to develop a set of molecular markers to investigate genetic diversity and population structure of E. ulmoides. Pyrosequencing of an enriched microsatellite library by Roche 454 FLX+ platform was used to isolate simple sequence repeats (SSRs) for E. ulmoides. A total of 1568 SSRs that contained enough flanking sequences for primer pair design were identified from 45,236 raw sequence reads. One hundred SSRs were randomly selected to design primer pairs and polymerase chain reaction was performed. Among these 100 tested primer pairs, 16 were polymorphic across 18 individuals from three E. ulmoides populations. The number of alleles ranged from 3 to 8, with an average of 5.1. The expected heterozygosity ranged from 0.110 to 0.830, with an average of 0.648, and the observed heterozygosity ranged from 0.111 to 0.833, with an average of 0.524. The inbreeding coefficient ranged from -0.349 to 0.547. This set of microsatellite markers could be valuable for landscape genetic structure assessment and molecular marker-assisted breeding in E. ulmoides.

Genetic diversity and population structure of Eucommia ulmoides Oliver, an endangered medicinal plant in China.

Eucommia ulmoides Oliver, one of the tertiary relict species found only in China, is the only extant species of Eucommiaceae. Using inter-simple sequence repeat (ISSR) and sequence-related amplified polymorphism (SRAP) markers, we studied the genetic diversity and population genetic structure of 187 accessions from 17 E. ulmoides populations throughout its main distribution in China. A total of 65 bands were amplified using eight ISSR primers, of which 50 bands (76.9%) were polymorphic. Meanwhile, another 244 bands were observed using eight SRAP primer combinations and 163 (66.8%) of these were polymorphic. The analysis of molecular variation (AMOVA) indicated that 88.8 and 92.4% of the total variation resided within populations based on ISSR and SRAP analysis, respectively. Moreover, we found that the E. ulmoides populations were clustered into six distinct groups using ISSR and SRAP markers via the unweighted pair-group method (UPGMA). Furthermore, STRUCTURE analysis showed that these 17 populations could be classified into four groups using ISSR markers, but only two groups using SRAP markers. No significant relevancy was observed between genetic and geographic distances among the sampled populations. The results of this study support the view that exchange of seeds among local farmers plays an important role in shaping the present genetic distribution pattern. "Core collection" is suggested for genetic diversity conservation of E. ulmoides in China.

Genetic diversity in intraspecific hybrid populations of Eucommia ulmoides Oliver evaluated from ISSR and SRAP molecular marker analysis.

Eucommia ulmoides Oliver, the only extant species of Eucommiaceae, is a second-category state-protected endangered plant in China. Evaluation of genetic diversity among some intraspecific hybrid populations of E. ulmoides Oliver is vital for breeding programs and further conservation of this rare species. We studied the genetic diversity of 130 accessions from 13 E. ulmoides intraspecific hybrid populations using inter-simple sequence related (ISSR) and sequence-related amplified polymorphism (SRAP) markers. Of the 100 ISSR primers and 100 SRAP primer combinations screened, eight ISSRs and eight SRAPs were used to evaluate the level of polymorphism and discriminating capacity. A total number of 65 bands were amplified using eight ISSR primers, in which 50 bands (76.9%) were polymorphic, with an average of 8.1 polymorphic fragments per primer. Alternatively, another 244 bands were observed using eight SRAP primer combinations, and 163 (66.8%) of them were polymorphic, with an average of 30.5 polymorphic fragments per primer. The unweighted pair-group method (UPGMA) analysis showed that these 13 populations could be classified into three groups by the ISSR marker and two groups by the SRAP marker. Principal coordinate analysis using SRAP was completely identical to the UPGMA-based clustering, although this was partly confirmed by the results of UPGMA cluster analysis using the ISSR marker. This study provides insights into the genetic background of E. ulmoides intraspecific hybrids. The progenies of the variations "Huazhong-3", "big fruit", "Yanci", and "smooth bark" present high genetic diversity and offer great potential for E. ulmoides breeding and conservation.

Quantitative trait locus analysis of leaf morphological characters, yield-related traits, and secondary metabolite contents in Eucommia ulmoides.

Eucommia ulmoides is cultivated for the production of eucommia rubber and Chinese herbal drugs. Molecular breeding methods, such as marker-assisted selection (MAS), have the potential to improve the efficiency of E. ulmoides breeding. Quantitative trait locus (QTL) analysis was applied to identify marker-trait associations for E. ulmoides using an F1 mapping population of 152 individuals derived from a cross between the wild genotype Xiaoye and the cultivar Qinzhong No. 1. A total of 78 QTLs were identified for 12 leaf traits involving morphology, yield, and secondary metabolites. Phenotypic variance explained by individual QTLs ranged from 10.4 to 53.3%. Fifteen QTL clusters, each harboring loci controlling at least two leaf traits, were detected across nine linkage groups. Co-location of these QTLs may be due to pleiotropy or linkage. Three main QTL regions for secondary metabolites were consistently identified each year. QTL information from this study furthers our understanding of the genetic architecture of these economically important traits and of MAS in E. ulmoides breeding.

[Specific PCR Molecular Identification of Euconunmia Cortex].

OBJECTIVE: To screen specific DNA sequence by rDNA-ITS sequence analysis at Eucommmia Cortex and its adulterants, then specific primers were designed to identify Eucommmia Cortex rapidly. METHODS: rDNA-ITS sequences of Eucommmia Cortex and its adulterants were downloaded from NCBI. The specific DNA sequence in the rDNA-ITS sequence of Eucommmia Cortex was found by compared with all sequences. Specific primers for authentic the Eucommmia Cortex were designed. PCR amplification and detection methods were optimized. RESULTS: PCR amplification products were detected through agarose gel electrophoresis and fluorescence reaction. The 400 bp identification band for Eucommmia Cortex could be produced and green fluorescent could be observed under ultraviolet light after adding the dye SYBR Green I. However its adulterants were without identification hand and green fluorescent. CONCLUSION: Specific PCR molecular identification can be applied to identify Eucommmia Cortex, the time is shortened significantly by PCR amplification optimization, and fluorescence detection can be more visually display the identification results. In a word, this study provides new ideas and methods for rapid identification of Eucommmia Cortex.

Relationship between progeny growth performance and molecular marker-based genetic distances in Eucommia ulmoides parental genotypes.

Progeny performances, variations and combining abilities for growth traits were evaluated in a factorial mating design of Eucommia ulmoides. Three marker systems, sequence-related amplified polymorphism, amplified fragment length polymorphism, and inter-simple sequence repeat, were used to determine genetic distances between parents. Correlations of genetic distances with progeny performances, within-family coefficients of variation and specific-combining abilities were established for height and basal diameter traits. Significant positive correlations were found between progeny performances of growth traits and genetic distances of parents based on sequence-related amplified polymorphism markers or a combination of all 3 marker systems. This revealed that crosses between genetically distant parents produced progenies with excellent growth performances. The lack of correlations between parental genetic distances and within-family coefficients of variation or specific-combining abilities suggested that these characteristics were unpredictable. The results of this study represent a potential criterion to predict progeny performances and choose parents in the breeding program.

Genetic diversity and relationships among Chinese Eucommia ulmoides cultivars revealed by sequence-related amplified polymorphism, amplified fragment length polymorphism, and inter-simple sequence repeat markers.

Sequence-related amplified polymorphism (SRAP), amplified fragment length polymorphism (AFLP), and inter-simple sequence repeat (ISSR) markers were used to estimate the genetic diversity and relationships among Eucommia ulmoides cultivars in China. A total of 240, 192, and 150 DNA fragments were detected by 10 SRAP primer combinations, 10 AFLP primer combinations, and 10 ISSR primers, among which 89.2, 65.1, and 88.0% of the fragments were polymorphic, respectively. Cluster analysis revealed that Qinzhong No. 3, Xiaoyeci, Qinzhong No. 1, and Qinzhong No. 2 formed independent clusters. The other 15 cultivars exhibited two clusters. The results of this study will help in the selection of parents for both genome mapping and crossbreeding purposes.