Characterization of chloroplast genome and phylogenetic analysis of the Elaeagnus hybrid variety E. 'viridis' (Elaeagnaceae).

Elaeagnus 'viridis', an artificial hybrid of E. macrophylla (♂) Thunb. (1784) × E. pungens (♀) Thunb. (1784), is known for its economic and ecological value. In this study, we sequenced and assembled the whole chloroplast (cp) genome of E. 'viridis'. The results showed that its cp genome was 152,284 bp long, showing a typical quadripartite structure and containing a large single-copy region (LSC, 82,299 bp), a small single-copy region (SSC, 18,239 bp), and a pair of inverted repeats (IRs, 51,746 bp). The cp genome contains 132 genes, including 86 protein-coding genes (PCGs), 38 tRNA genes, and 8 rRNA genes. Phylogenetic analysis based on 66 common PCGs revealed that E. 'viridis' is most closely related to its maternal parent E. pungens. The chloroplast genomic information reported in this study will shed some useful light for further genetic studies in the genus Elaeagnus.

Complete chloroplast genome of Ilex dabieshanensis: Genome structure, comparative analyses with three traditional Ilex tea species, and its phylogenetic relationships within the family Aquifoliaceae.

Ilex dabieshanensis K. Yao & M. B. Deng is not only a highly valued tree species for landscaping, it is also a good material for making kuding tea due to its anti-inflammatory and lipid-lowering medicinal properties. Utilizing next-generation and long-read sequencing technologies, we assembled the whole chloroplast genome of I. dabieshanensis. The genome was 157,218 bp in length, exhibiting a typical quadripartite structure with a large single copy (LSC: 86,607 bp), a small single copy (SSC: 18,427 bp) and a pair of inverted repeat regions (IRA and IRB: each of 26,092 bp). A total of 121 predicted genes were encoded, including 113 distinctive (79 protein-coding genes, 30 tRNAs, and 4 rRNAs) and 8 duplicated (8 protein-coding genes) located in the IR regions. Overall, 132 SSRs and 43 long repeats were detected and could be used as potential molecular markers. Comparative analyses of four traditional Ilex tea species (I. dabieshanensis, I. paraguariensis, I. latifolia and I. cornuta) revealed seven divergent regions: matK-rps16, trnS-psbZ, trnT-trnL, atpB-rbcL, petB-petD, rpl14-rpl16, and rpl32-trnL. These variations might be applicable for distinguishing different species within the genus Ilex. Phylogenetic reconstruction strongly suggested that I. dabieshanensis formed a sister clade to I. cornuta and also showed a close relationship to I. latifolia. The generated chloroplast genome information in our study is significant for Ilex tea germplasm identification, phylogeny and genetic improvement.

Agrobacterium-Mediated Genetic Transformation of Embryogenic Callus in a Liriodendron Hybrid (L. Chinense × L. Tulipifera).

A highly efficient genetic transformation system of Liriodendron hybrid embryogenic calli through Agrobacterium-mediated genetic transformation was established and optimized. The Agrobacterium tumefaciens strain EHA105, harboring the plasmid pBI121, which contained the ß-glucuronidase (GUS) gene and neomycin phosphotransferase II (npt II) gene under the control of the CaMV35S promoter, was used for transformation. Embryogenic calli were used as the starting explant to study several factors affecting the Agrobacterium-mediated genetic transformation of the Liriodendron hybrid, including the effects of various media, selection by different Geneticin (G418) concentrations, pre-culture period, Agrobacterium optical density, infection duration, co-cultivation period, and delayed selection. Transformed embryogenic calli were obtained through selection on medium containing 90 mg L-1 G418. Plant regeneration was achieved and selected via somatic embryogenesis on medium containing 15 mg L-1 G418. The optimal conditions included a pre-culture time of 2 days, a co-culture time of 3 days, an optimal infection time of 10 min, and a delayed selection time of 7 days. These conditions, combined with an OD600 value of 0.6, remarkably enhanced the transformation rate. The results of GUS chemical tissue staining, polymerase chain reaction (PCR), and southern blot analysis demonstrated that the GUS gene was successfully expressed and integrated into the Liriodendron hybrid genome. A transformation efficiency of 60.7% was achieved for the regenerated callus clumps. Transgenic plantlets were obtained in 5 months, and the PCR analysis showed that 97.5% of plants from the tested G418-resistant lines were PCR positive. The study of the Liriodendron hybrid reported here will facilitate the insertion of functional genes into the Liriodendron hybrid via Agrobacterium-mediated transformation.

Characterization and phylogenetic analysis of the complete chloroplast genome of Ilex rotunda, traditional Chinese medicine plant.

Ilex rotunda is a traditional Chinese medicine plant. In this study, we characterized the complete chloroplast (cp) genome sequence of I. rotunda to investigate its phylogenetic relationship. The cp genome of I. rotunda was 157,743 bp in length with 37.62% overall GC content, including a large single-copy (LSC) region of 87,060bp, a small single-copy (SSC) region of 18,432 bp, which were separated by a pair of inverted repeats (IRS) of 26,121 bp. The cp genome contained 133 genes, including 88 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Phylogenetic analysis based on whole cp genome sequences showed that I. rotunda is closely related to I. pubescens and I. polyneura.

Complete chloroplast genome sequence and phylogenetic analysis of Ilex × attenuata 'Fosteri' (Aquifoliaceae).

Ilex × attenuata 'Fosteri' is an important ornamental plant widely distributed in mid-southern China and south-eastern United States. In this study, we assembled the complete chloroplast (cp) genome of I. attenuata by high-throughput sequencing and bioinformatics. The full length of cp genome was 157,833 bp with 37.63% overall GC content, which contained two inverted repeats (IR) of 26,093 bp separated by a large single-copy (LSC) and a small single copy (SSC) of 87,188 bp and 18,459 bp, respectively. The cp genome contained 135 genes, including 88 protein-coding genes, 8 rRNA genes and 39 tRNA genes. Phylogenetic tree showed that the close relationship of three species of Ilex (I. attenuata, I. viridis and I. szechwanensis) in the Aquifoliaceae family.

Characterization of the complete chloroplast genome of Ilex crenata Thunb. (Aquifoliaceae).

Ilex crenata Thunb. is a species of Aquifoliaceae with high ornamental and ecological values. In this study, the complete chloroplast (cp) genome of I. crenata was assembled and characterized through Illumina sequencing data. The entire cp genome of I. crenata was 157,988 bp in length with 37.64% overall GC content, containing a large single-copy (LSC) region of 87,414 bp and a small single-copy (SSC) region of 18,422 bp, which were separated by a pair of 26,076 bp inverted repeat (IR) regions. A total of 135 genes were annotated, including 88 protein-coding genes, 39 tRNA genes, and 8 rRNA genes. Phylogenetic analysis based on 78 conserved protein-coding genes demonstrated that I. crenata is closely related to I. viridis and I. szechwanensis.

Integrative analysis of transcriptome and proteome revealed nectary and nectar traits in the plant-pollinator interaction of Nitraria tangutorum Bobrov.

BACKGROUND: Nitraria tangutorum is an important desert shrub that shows resistance to drought, salt and wind erosion stresses. It is a central ecological species in its area. Here, we have studied how N. tangutorum has adapted to achieve a successful reproduction strategy. RESULTS: We found that N. tangutorum is mainly pollinated by insects of the Hymenoptera, Diptera and Coleoptera orders. Nitraria tangutorum has very small flowers, with the nectary composed of secretive epidermal cells from which nectar is secreted, located within the inner petals. In addition, analyzing the transcriptome of four successive flower developmental stages revealed that mainly differentially expressed genes associated with flower and nectary development, nectar biosynthesis and secretion, flavonoid biosynthesis, plant hormone signal transduction and plant-pathogen interaction show dynamic expression. From the nectar, we could identify seven important proteins, of which the L-ascorbate oxidase protein was first found in plant nectar. Based on the physiological functions of these proteins, we predict that floral nectar proteins of N. tangutorum play an important role in defending against microbial infestation and scavenging active oxygen. CONCLUSIONS: This study revealed that N. tangutorum is an insect-pollinated plant and its nectary is composed of secretive epidermal cells that specialized into secretive trichomes. We identified a large number of differentially expressed genes controlling flower and nectary development, nectar biosynthesis and secretion, flavonoid biosynthesis, plant hormone signal transduction and plant-pathogen interaction. We suggest that proteins present in N. tangutorum nectar may have both an antibacterial and oxygen scavenging effect. These results provide a scientific basis for exploring how the reproductive system of N. tangutorum and other arid-desert plants functions.

The complete chloroplast genome sequence of Taxodium ascendens × T. mucronatum hybrid (Cupressaceae).

Taxodium 'Zhongshanshan 401' is an important economic plant with ornamental and ecological values, and has been widely planted in southeastern China. In this study, the complete chloroplast (cp) genome of T. 'Zhongshanshan 401' was sequenced and illustrated to add the more genetic information. The entire cp genome of T. 'Zhongshanshan 401' was 132,037 bp in length with 35.3% overall GC content. The cp genome contained 120 genes, including 83 protein-coding genes, 33 tRNA genes, and four rRNA genes. Fifteen genes contain two exons and two contains three exons. Phylogenetic analysis based on whole cp genome sequences showed that T. 'Zhongshanshan 401' was more closely related to T. mucronatum.

The complete chloroplast genome sequence of Ilex chinensis Sims. (Aquifoliaceae), a folk herbal medicine plant in China.

The complete chloroplast (cp) genome of Ilex chinensis, an important economic plant with ornamental and ecological values, was sequenced to investigate its phylogenetic relationship. The entire cp genome of I. chinensis was 157,885 bp in length with 37.61% overall GC content, including a large single-copy (LSC) region of 87,289 bp, and a small single-copy (SSC) region of 18,388 bp, which were separated by a pair of inverted repeats (IRs) of 52,208 bp. The cp genome contained 135 genes, including 90 protein-coding genes, 37 tRNA genes, and eight rRNA genes. Phylogenetic analysis based on whole cp genome sequences showed that I. chinensis was closely related to I. szechwanensis and I. viridis species.

The complete chloroplast genome sequence and phylogenetic analysis of Ilex × Koehneana 'Wirt L. Winn' (Aquifoliaceae).

Ilex × Koehneana 'Wirt L. Winn', an important ornamental tree, has been widely distributed in southeastern China. In this study, we assembled and characterized the complete chloroplast (cp) genome of I. Koehneana to investigate its phylogenetic relationship. The whole cp genome of I. Koehneana is 157,538 bp, which contained a large single-copy (LSC) region of 87,055 bp and a small single-copy (SSC) region of 18,429 bp, and a pair of inverted repeats (IR) of 52,054 bp. A total of 137 genes, including 90 protein-coding genes, eight rRNAs, and 39 tRNAs, were identified. Phylogenetic analysis based on 74 conserved protein-coding genes revealed that I. Koehneana is closely related to I. 'tall boy'.

The complete chloroplast genome sequence and phylogenetic analysis of Ilex 'Beryl', a hybrid of Ilex cornuta × Ilex latifolia (Aquifoliaceae).

Ilex 'Beryl' is an ornamental and ecological tree widespread in southeastern China. In this study, the complete chloroplast (cp) genome of Ilex 'Beryl' was assembled and characterized to investigate its phylogenetic relationship. The entire cp genome of 'Beryl' was a typical quadripartite structure with 157,575 bp in length, including a large single-copy (LSC) region of 87,080 bp and a small single-copy (SSC) region of 18,427 bp, which were separated by a pair of inverted repeats (IRs) of 52,068 bp. There are 135 genes annotated, including 90 protein-coding genes, eight rRNA genes and 37 tRNA genes. Phylogenetic analysis based on whole cp genome sequences showed that 'Beryl' is closest to I. 'Emily Bruner' and I. 'tall boy'.

The complete chloroplast genome of Ilex 'Tall Boy', Ilex aquifolium × Ilex latifolia (Aquifoliaceae).

Thecomplete chloroplast (cp) genome of Ilex 'Tall Boy', an important economic plant with ornamental and ecological values, was sequenced to investigate its phylogenetic relationship. The entire cp genome of 'Tall Boy' was 157,527 bp in length with 37.65% overall GC content, including a large single-copy (LSC) region of 87,044 bp and a small single-copy (SSC) region of 18,429 bp, which were separated by a pair of inverted repeats (IRs) of 52,054 bp. The cp genome contained 135 genes, including 90 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Phylogenetic analysis based on whole cp genome sequences showed that 'Tall Boy' is closest to I. latifolia Thunb. species.

The complete chloroplast genome of Ilex 'Emily Bruner', Ilex cornuta 'Burfordii' × Ilex latifolia (Aquifoliaceae).

Ilex 'Emily Bruner' is an important economic plant with ornamental and ecological functions in southeastern China. In this study, we characterized the complete chloroplast (cp) genome sequence of 'Emily Bruner' to investigate its phylogenetic relationship. The entire cp genome of 'Emily Bruner' was 157,216 bp in length with 37.68% overall GC content, including a large single-copy (LSC) region of 86,721 bp and a small single-copy (SSC) region of 18,427 bp, which were separated by a pair of inverted repeats (IRs) of 52,068 bp. The cp genome contained 135 genes, including 90 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Phylogenetic analysis based on whole cp genome sequences showed that 'Emily Bruner' is closest to I. cornuta species.

Author Correction: Liriodendron genome sheds light on angiosperm phylogeny and species-pair differentiation.

In the Supplementary Information file originally published with this Letter, the authors mistakenly omitted Supplementary Table 14; this has now been amended.

Liriodendron genome sheds light on angiosperm phylogeny and species-pair differentiation.

The genus Liriodendron belongs to the family Magnoliaceae, which resides within the magnoliids, an early diverging lineage of the Mesangiospermae. However, the phylogenetic relationship of magnoliids with eudicots and monocots has not been conclusively resolved and thus remains to be determined1-6. Liriodendron is a relict lineage from the Tertiary with two distinct species-one East Asian (L. chinense (Hemsley) Sargent) and one eastern North American (L. tulipifera Linn)-identified as a vicariad species pair. However, the genetic divergence and evolutionary trajectories of these species remain to be elucidated at the whole-genome level7. Here, we report the first de novo genome assembly of a plant in the Magnoliaceae, L. chinense. Phylogenetic analyses suggest that magnoliids are sister to the clade consisting of eudicots and monocots, with rapid diversification occurring in the common ancestor of these three lineages. Analyses of population genetic structure indicate that L. chinense has diverged into two lineages-the eastern and western groups-in China. While L. tulipifera in North America is genetically positioned between the two L. chinense groups, it is closer to the eastern group. This result is consistent with phenotypic observations that suggest that the eastern and western groups of China may have diverged long ago, possibly before the intercontinental differentiation between L. chinense and L. tulipifera. Genetic diversity analyses show that L. chinense has tenfold higher genetic diversity than L. tulipifera, suggesting that the complicated regions comprising east-west-orientated mountains and the Yangtze river basin (especially near 30° N latitude) in East Asia offered more successful refugia than the south-north-orientated mountain valleys in eastern North America during the Quaternary glacial period.

Desiccation Treatment and Endogenous IAA Levels Are Key Factors Influencing High Frequency Somatic Embryogenesis in Cunninghamia lanceolata (Lamb.) Hook.

Cunninghamia lanceolata (Lamb.) Hook (Chinese fir) is an important tree, commercially and ecologically, in southern China. The traditional regenerating methods are based on organogenesis and cutting propagation. Here, we report the development of a high-frequency somatic embryogenesis (SE) regeneration system synchronized via a liquid culture from immature zygotic embryos. Following synchronization, PEM II cell aggregates were developmentally equivalent in appearance to cleaved zygotic embryos. Embryo and suspensor growth and subsequent occurrence of the apical and then the cotyledonary meristems were similar for zygotic and SE embryo development. However, SE proembryos exhibited a more reddish coloration than zygotic proembryos, and SE embryos were smaller than zygotic embryos. Mature somatic embryos gave rise to plantlets on hormone-free medium. For juvenile explants, low concentrations of endogenous indole-3-acetic acid in initial explants correlated with improved proembryogenic mass formation, and high SE competency. Analysis of karyotypes and microsatellites detected no major genetic variation in the plants regenerated via SE, and suggest a potential in the further development of this system as a reliable methodology for true-to-type seedling production. Treatment with polyethylene glycol (PEG) and abscisic acid (ABA) were of great importance to proembryo formation and complemented each other. ABA assisted the growth of embryonal masses, whereas PEG facilitated the organization of the proembryo-like structures. SOMATIC EMBRYOGENESIS RECEPTOR KINASE SERK) and the WUSCHEL homeobox (WOX) transcription factor served as molecular markers during early embryogenesis. Our results show that ClSERKs are conserved and redundantly expressed during SE. SERK and WOX transcript levels were highest during development of the proembryos and lowest in developed embryos. ClWOX13 expression correlates with the critical transition from proembryogenic masses to proembryos. Both SERK and WOX expression reveal their applicability in Chinese fir as markers of early embryogenesis. Overall, the findings provided evidence for the potential of this system in high fidelity Chinese fir seedlings production. Also, SE modification strategies were demonstrated and could be applied in other conifer species on the basis of our hormonal, morphological and molecular analyses.

Ground transport stress affects bacteria in the rumen of beef cattle: A real-time PCR analysis.

Transport stress syndrome often appears in beef cattle during ground transportation, leading to changes in their capacity to digest food due to changes in rumen microbiota. The present study aimed to analyze bacteria before and after cattle transport. Eight Xianan beef cattle were transported over 1000 km. Rumen fluid and blood were sampled before and after transport. Real-time PCR was used to quantify rumen bacteria. Cortisol and adrenocorticotrophic hormone (ACTH) were measured. Cortisol and ACTH were increased on day 1 after transportation and decreased by day 3. Cellulolytic bacteria (Fibrobacter succinogenes and Ruminococcus flavefaciens), Ruminococcus amylophilus and Prevotella albensis were increased at 6 h and declined by 15 days after transport. There was a significant reduction in Succinivibrio dextrinosolvens, Prevotella bryantii, Prevotella ruminicola and Anaerovibrio lipolytica after transport. Rumen concentration of acetic acid increased after transport, while rumen pH and concentrations of propionic and butyric acids were decreased. Body weight decreased by 3 days and increased by 15 days after transportation. Using real-time PCR analysis, we detected changes in bacteria in the rumen of beef cattle after transport, which might affect the growth of cattle after transport.

The Complete Chloroplast Genome Sequence of a Relict Conifer Glyptostrobus pensilis: Comparative Analysis and Insights into Dynamics of Chloroplast Genome Rearrangement in Cupressophytes and Pinaceae.

Glyptostrobus pensilis, belonging to the monotypic genus Glyptostrobus (Family: Cupressaceae), is an ancient conifer that is naturally distributed in low-lying wet areas. Here, we report the complete chloroplast (cp) genome sequence (132,239 bp) of G. pensilis. The G. pensilis cp genome is similar in gene content, organization and genome structure to the sequenced cp genomes from other cupressophytes, especially with respect to the loss of the inverted repeat region A (IRA). Through phylogenetic analysis, we demonstrated that the genus Glyptostrobus is closely related to the genus Cryptomeria, supporting previous findings based on physiological characteristics. Since IRs play an important role in stabilize cp genome and conifer cp genomes lost different IR regions after splitting in two clades (cupressophytes and Pinaceae), we performed cp genome rearrangement analysis and found more extensive cp genome rearrangements among the species of cupressophytes relative to Pinaceae. Additional repeat analysis indicated that cupressophytes cp genomes contained less potential functional repeats, especially in Cupressaceae, compared with Pinaceae. These results suggested that dynamics of cp genome rearrangement in conifers differed since the two clades, Pinaceae and cupressophytes, lost IR copies independently and developed different repeats to complement the residual IRs. In addition, we identified 170 perfect simple sequence repeats that will be useful in future research focusing on the evolution of genetic diversity and conservation of genetic variation for this endangered species in the wild.

Floral Nectary Morphology and Proteomic Analysis of Nectar of Liriodendron tulipifera Linn.

Nectar is a primary nutrient reward for a variety of pollinators. Recent studies have demonstrated that nectar also has defensive functions against microbial invasion. In this study, the Liriodendron tulipifera nectary was first examined by scanning electron microscopy, and then the nectar was analyzed by two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry, which led to identification of 42 nectar proteins involved in various biological functions. Bioinformatic analysis was then performed on an identified novel rubber elongation factor (REF) protein in L. tulipifera nectar. The protein was particularly abundant, representing ∼60% of the major bands of 31 to 43 kDa, and showed high, stage-specific expression in nectary tissue. The REF family proteins are the major allergens in latex. We propose that REF in L. tulipifera nectar has defensive characteristics against microorganisms.

Physiological and proteomic analyses of leaves from the halophyte Tangut Nitraria reveals diverse response pathways critical for high salinity tolerance.

Soil salinization poses a serious threat to the environment and agricultural productivity worldwide. Studies on the physiological and molecular mechanisms of salinity tolerance in halophytic plants provide valuable information to enhance their salt tolerance. Tangut Nitraria is a widely distributed halophyte in saline-alkali soil in the northern areas of China. In this study, we used a proteomic approach to investigate the molecular pathways of the high salt tolerance of T. Nitraria. We analyzed the changes in biomass, photosynthesis, and redox-related enzyme activities in T. Nitraria leaves from plant seedlings treated with high salt concentration. Comparative proteomic analysis of the leaves revealed that the expression of 71 proteins was significantly altered after salinity treatments of T. Nitraria. These salinity-responsive proteins were mainly involved in photosynthesis, redox homeostasis, stress/defense, carbohydrate and energy metabolism, protein metabolism, signal transduction, and membrane transport. Results showed that the reduction of photosynthesis under salt stress was attributed to the down-regulation of the enzymes and proteins involved in the light reaction and Calvin cycle. Protein-protein interaction analysis revealed that the proteins involved in redox homeostasis, photosynthesis, and energy metabolism constructed two types of response networks to high salt stress. T. Nitraria plants developed diverse mechanisms for scavenging reactive oxygen species (ROS) in their leaves to cope with stress induced by high salinity. This study provides important information regarding the salt tolerance of the halophyte T. Nitraria.