CmDOF18 positively regulates salinity tolerance in Chrysanthemum morifolium by activating the oxidoreductase system.

BACKGROUND: Chrysanthemum, one of the four major cut flowers all over the world, is very sensitive to salinity during cultivation. DNA binding with one finger (DOF) transcription factors play important roles in biological processes in plants. The response mechanism of CmDOF18 from chrysanthemum to salt stress remains unclear. RESULTS: In this study, CmDOF18 was cloned from Chrysanthemum morifolium, and its expression was induced by salinity stress. The gene encodes a 291-amino acid protein with a typical DOF domain. CmDOF18 was localized to the nucleus in onion epidermal cells and showed transcriptional activation in yeast. CmDOF18 transgenic plants were generated to identify the role of this gene in resistance to salinity treatment. Chrysanthemum plants overexpressing CmDOF18 were more resistant to salinity stress than wild-type plants. Under salinity stress, the malondialdehyde content and leaf electrolyte conductivity in CmDOF18-overexpressing transgenic plants were lower than those in wild-type plants, while the proline content, chlorophyll content, superoxide dismutase activity and peroxidase activity were higher than those in wild-type plants. The opposite findings were observed in gene-silenced plants compared with wild-type plants. The gene expression levels of oxidoreductase increased in CmDOF18-overexpressing transgenic plants but decreased in CmDOF18-SRDX gene-silenced transgenic plants. CONCLUSION: In summary, we analyzed the function of CmDOF18 from chrysanthemum, which may regulate salinity stress in plants, possibly due to its role in the regulation of oxidoreductase.

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.

Novel molecular markers for Taxodium breeding from the chloroplast genomes of four artificial Taxodium hybrids.

Taxodium "Zhongshanshan" are a group of intraspecific Taxodium hybrids with superparental dominance and high ecological and economic value in southern China. Identifying the parentage of hybrids, especially the male parent, is critically important for genetic studies. However, the large nuclear genomes of members of the genus Taxodium pose a major challenge for the development of molecular markers. Here, we developed novel molecular markers by conducting a comparative analysis of the chloroplast genomes of four artificial Taxodium hybrids and their parents. The lengths of the whole chloroplast genome ranged from 131,942 to 132,128 bp, and the total guanine (GC) content of the chloroplast genomes ranged from 34.6% to 35.81%. A total of 120 unique genes were identified, including 83 protein-coding genes, 33 transfer RNAs, and four ribosomal RNAs. There were 69-71 simple sequence repeats were detected in the four hybrids. Phylogenetic analysis revealed that these hybrids clustered with their paternal parents. Similar findings were obtained by analysis of the GC content of protein-coding genes. Molecular markers were developed using the highly variable regions of the chloroplast genomes, and polymerase chain reaction (PCR) assays revealed that these markers were effective for identifying the male parents of these hybrids. Our findings indicate for the first time that the chloroplast genomes of Taxodium are paternally inherited. Generally, these molecular markers could facilitate breeding and genetic studies of Taxodium.

Flowering repressor CmSVP recruits the TOPLESS corepressor to control flowering in chrysanthemum.

Plant flowering time is induced by environmental and endogenous signals perceived by the plant. The MCM1-AGAMOUSDEFICIENS-Serum Response Factor-box (MADS-box) protein SHORT VEGETATIVE PHASE (SVP) is a pivotal repressor that negatively regulates the floral transition during the vegetative phase; however, the transcriptional regulatory mechanism remains poorly understood. Here, we report that CmSVP, a chrysanthemum (Chrysanthemum morifolium Ramat.) homolog of SVP, can repress the expression of a key flowering gene, a chrysanthemum FLOWERING LOCUS T-like gene (CmFTL3), by binding its promoter CArG element to delay flowering in the ambient temperature pathway in chrysanthemum. Protein-protein interaction assays identified an interaction between CmSVP and CmTPL1-2, a chrysanthemum homologue of TOPLESS (TPL) that plays critical roles as transcriptional corepressor in many aspects of plant life. Genetic analyses revealed the CmSVP-CmTPL1-2 transcriptional complex is a prerequisite for CmSVP to act as a floral repressor. Furthermore, overexpression of CmSVP rescued the phenotype of the svp-31 mutant in Arabidopsis (Arabidopsis thaliana), overexpression of AtSVP or CmSVP in the Arabidopsis dominant-negative mutation tpl-1 led to ineffective late flowering, and AtSVP interacted with AtTPL, confirming the conserved function of SVP in chrysanthemum and Arabidopsis. We have validated a conserved machinery wherein SVP partially relies on TPL to inhibit flowering via a thermosensory pathway.

Comprehensive Genomic Analysis of SnRK in Rosaceae and Expression Analysis of RoSnRK2 in Response to Abiotic Stress in Rubus occidentalis.

The sucrose nonfermenting 1-related protein kinase (SnRK) plays an important role in responding to abiotic stresses by phosphorylating the target protein to regulate various signaling pathways. However, little is known about the characteristics, evolutionary history, and expression patterns of the SnRK family in black raspberry (Rubus occidentalis L.) or other Rosaceae family species. In this study, a total of 209 SnRK genes were identified in 7 Rosaceae species and divided into 3 subfamilies (SnRK1, SnRK2, and SnRK3) based on phylogenetic analysis and specific motifs. Whole-genome duplication (WGD) and dispersed duplication (DSD) were considered to be major contributions to the SnRK family expansion. Purifying selection was the primary driving force in the SnRK family evolution. The spatial expression indicated that the RoSnRK genes may play important roles in different tissues. In addition, the expression models of 5 RoSnRK2 genes in response to abiotic stresses were detected by qRT-PCR. The proteins encoded by RoSnRK2 genes localize to the cytoplasm and nucleus in order to perform their respective functions. Taken together, this study provided an analysis of the SnRK gene family expansion and evolution, and contributed to the current knowledge of the function of 5 RoSnRK2 genes, which in turn expanded understanding of the molecular mechanisms of black raspberry responses to abiotic stress.

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.

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.

Genome-Wide Identification and Expression Analysis of BBX Transcription Factors in Iris germanica L.

The family of B-box (BBX) transcription factors contains one or two B-BOX domains and sometimes also features a highly conserved CCT domain, which plays important roles in plant growth, development and stress response. Nevertheless, no systematic study of the BBX gene family in Iris germanica L. has been undertaken. In this study, a set of six BBX TF family genes from I. germanica was identified based on transcriptomic sequences, and clustered into three clades according to phylogenetic analysis. A transient expression analysis revealed that all six BBX proteins were localized in the nucleus. A yeast one-hybrid assay demonstrated that IgBBX3 has transactivational activity, while IgBBX1, IgBBX2, IgBBX4, and IgBBX5 have no transcriptional activation ability. The transcript abundance of IgBBXs in different tissues was divided into two major groups. The expression of IgBBX1, IgBBX2, IgBBX3 and IgBBX5 was higher in leaves, whereas IgBBX4 and IgBBX6 was higher in roots. The stress response patterns of six IgBBX were detected under phytohormone treatments and abiotic stresses. The results of this study lay the basis for further research on the functions of BBX gene family members in plant hormone and stress responses, which will promote their application in I. germanica breeding.

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'.

Heterologous expression of chrysanthemum TOPLESS corepressor CmTPL1-1 alters meristem maintenance and organ development in Arabidopsis thaliana.

TOPLESS (TPL)/TOPLESS-related (TPR) corepressors are important regulators of plant growth and development, but their functions in chrysanthemum (Chrysanthemum morifolium) are currently unclear. In this study, a chrysanthemum TPL/TPR family gene, designated CmTPL1-1, was characterized. This gene encodes an 1135-amino-acid polypeptide harboring a conserved N-terminal domain and two C-terminal WD40 domains. CmTPL1-1 showed no transcriptional activity in yeast, and a localization experiment indicated that it localized to the nuclei in onion epidermal cells. Transcript profiling established that the gene was most highly expressed in the stem apex. The heterologous expression of CmTPL1-1 in Arabidopsis thaliana produced a pleiotropic phenotype, including smaller leaves, shorter siliques, increased meristem number, asymmetrical petal distribution and reduced stamen number. In transgenic plants, four AtARFs were downregulated, while six AtIAAs and two AtGH3s were upregulated at the transcript level; moreover, the expression of three key class I KNOTTED-like homeobox (KNOX) genes was upregulated. In addition, by yeast two-hybrid screening of a chrysanthemum cDNA library, we found that CmTPL1-1 could interact with CmWOX4, CmLBD38 and CmLBD36, and these interactions were confirmed by bimolecular fluorescence complementation (BiFC) assays. Overall, we speculated that heterologous expression of CmTPL1-1 regulates plant growth and development by interacting with auxin signaling in Arabidopsis.

Genome-wide association study identifies favorable SNP alleles and candidate genes for waterlogging tolerance in chrysanthemums.

Chrysanthemums are sensitive to waterlogging stress, and the development of screening methods for tolerant germplasms or genes and the breeding of tolerant new varieties are of great importance in chrysanthemum breeding. To understand the genetic basis of waterlogging tolerance (WT) in chrysanthemums, we performed a genome-wide association study (GWAS) using 92,811 single nucleotide polymorphisms (SNPs) in a panel of 88 chrysanthemum accessions, including 64 spray cut and 24 disbud chrysanthemums. The results showed that the average MFVW (membership function value of waterlogging) of the disbud type (0.65) was significantly higher than that of the spray type (0.55) at P < 0.05, and the MFVW of the Asian accessions (0.65) was significantly higher than that of the European accessions (0.48) at P < 0.01. The GWAS performed using the general linear model (GLM) and mixed linear model (MLM) identified 137 and 14 SNP loci related to WT, respectively, and 11 associations were commonly predicted. By calculating the phenotypic effect values for 11 common SNP loci, six highly favorable SNP alleles that explained 12.85-21.85% of the phenotypic variations were identified. Furthermore, the dosage-pyramiding effects of the favorable alleles and the significant linear correlations between the numbers of highly favorable alleles and phenotypic values were identified (r 2 = 0.45; P < 0.01). A major SNP locus (Marker6619-75) was converted into a derived cleaved amplified polymorphic sequence (dCAPS) marker that cosegregated with WT with an average efficiency of 78.9%. Finally, four putative candidate genes in the WT were identified via quantitative real-time PCR (qRT-PCR). The results presented in this study provide insights for further research on WT mechanisms and the application of molecular marker-assisted selection (MAS) in chrysanthemum WT breeding programs.

Identification of favorable SNP alleles and candidate genes responsible for inflorescence-related traits via GWAS in chrysanthemum.

KEY MESSAGE: 81 SNPs were identified for three inflorescence-related traits, in which 15 were highly favorable. Two dCAPS markers were developed for future MAS breeding, and six candidate genes were predicted. Chrysanthemum is a leading ornamental species worldwide and demonstrates a wealth of morphological variation. Knowledge about the genetic basis of its phenotypic variation for key horticultural traits can contribute to its effective management and genetic improvement. In this study, we conducted a genome-wide association study (GWAS) based on two years of phenotype data and a set of 92,617 single nucleotide polymorphisms (SNPs) using a panel of 107 diverse cut chrysanthemums to dissect the genetic control of three inflorescence-related traits. A total of 81 SNPs were significantly associated with the three inflorescence-related traits (capitulum diameter, number of ray florets and flowering time) in at least one environment, with an individual allele explaining 22.72-38.67% of the phenotypic variation. Fifteen highly favorable alleles were identified for the three target traits by computing the phenotypic effect values for the stable associations detected in 2 year-long trials at each locus. Dosage pyramiding effects of the highly favorable SNP alleles and significant linear correlations between highly favorable allele numbers and corresponding phenotypic performance were observed. Two highly favorable SNP alleles correlating to flowering time and capitulum diameter were converted to derived cleaved amplified polymorphic sequence (dCAPS) markers to facilitate future breeding. Finally, six putative candidate genes were identified that contribute to flowering time and capitulum diameter. These results serve as a foundation for analyzing the genetic mechanisms underlying important horticultural traits and provide valuable insights into molecular marker-assisted selection (MAS) in chrysanthemum breeding programs.

A SNP-Enabled Assessment of Genetic Diversity, Evolutionary Relationships and the Identification of Candidate Genes in Chrysanthemum.

Varieties of the economically important ornamental species chrysanthemum have been bred to fit a number of market niches, but the genetic basis and evolutionary relationships among various cultivated types are poorly understood. Here, a DNA marker-based analysis of 199 chrysanthemum entries representing each of the five cultivated types is presented. A set of >90,000 single nucleotide polymorphisms (SNPs) associated with a minor allele frequency of at least 5% was defined, and used to perform a phylogenetic analysis which corresponded well with the phenotypic classification. The analysis revealed that the small-flowered types, spray cut chrysanthemum (SCC) and potted and ground chrysanthemum (PGC), are more closely related to the wild progenitor species (WC) than are the large-flowered ones, disbud cut chrysanthemum (DCC) and traditional chrysanthemum (TC); and the PGC type was closest. Some 550 genetic regions appeared to have experienced selection in the separation of potted and ground-cover types from disbud cut types, and that between potted and ground-cover types from traditional types. A genome-wide association analysis revealed that seven SNPs lying within six genes were predictive of three important traits (ray floret type, cultivated type and flower shape), but no association with flower color was detected. The study has provided a number of novel insights into evolutionary relationships, the population structure and the genetic basis of some key ornamental traits.