Small RNA and Degradome Sequencing Reveal Roles of miRNAs in the Petal Color Fading of Malus Crabapple.

The Malus crabapple is an important woody ornamental plant. The fading of petals during its development significantly affects their ornamental value. Petal color is related to anthocyanin content and miRNAs play an important role in the post-transcriptional regulation of anthocyanin synthesis. However, the mechanisms underlying miRNA regulation of petal fading have rarely been studied. Transcriptome and small RNA sequencing of petals from the blooming phases of Malus. 'Indian Summer' varieties S1 (small bud), S2 (initial-flowering), and S3 (late-flowering) allowed us to identify 230 known miRNAs and 17 novel miRNAs, including 52 differentially expressed miRNAs which targeted 494 genes and formed 823 miRNA-target pairs. Based on the target gene annotation results, miRNA-target pairs were screened that may be involved in the fading process of Malus crabapple petals through three different pathways: anthocyanin synthesis, transport, and degradation, involving mcr-miR858-MYB1\MYB5 and mcr-miR396-McCHI inhibiting anthocyanin synthesis; mcr-miR167, mcr-miR390, mcr-miR535, and mcr-miR858 inhibiting anthocyanin transport from the cytoplasm to the vacuole by targeting ABC transporter genes (ABCB, ABCC, ABCD, and ABCG); and mcr-miR398 targeting the superoxide dismutase genes (CZSOD2 and CCS) to accelerate anthocyanin degradation. These findings offer a novel approach to understanding the mechanism of petal fading and serve as a reference for other plants with floral fading.

Gene Structural Specificity and Expression of MADS-Box Gene Family in Camellia chekiangoleosa.

MADS-box genes encode transcription factors that affect plant growth and development. Camellia chekiangoleosa is an oil tree species with ornamental value, but there have been few molecular biological studies on the developmental regulation of this species. To explore their possible role in C. chekiangoleosa and lay a foundation for subsequent research, 89 MADS-box genes were identified across the whole genome of C. chekiangoleosa for the first time. These genes were present on all the chromosomes and were found to have expanded by tandem duplication and fragment duplication. Based on the results of a phylogenetic analysis, the 89 MADS-box genes could be divided into either type I (38) or type II (51). Both the number and proportion of the type II genes were significantly greater than those of Camellia sinensis and Arabidopsis thaliana, indicating that C. chekiangoleosa type II genes experienced a higher duplication rate or a lower loss rate. The results of both a sequence alignment and a conserved motif analysis suggest that the type II genes are more conserved, meaning that they may have originated and differentiated earlier than the type I genes did. At the same time, the presence of extra-long amino acid sequences may be an important feature of C. chekiangoleosa. Gene structure analysis revealed the number of introns of MADS-box genes: twenty-one type I genes had no introns, and 13 type I genes contained only 1~2 introns. The type II genes have far more introns and longer introns than the type I genes do. Some MIKCC genes have super large introns (≥15 kb), which are rare in other species. The super large introns of these MIKCC genes may indicate richer gene expression. Moreover, the results of a qPCR expression analysis of the roots, flowers, leaves and seeds of C. chekiangoleosa showed that the MADS-box genes were expressed in all those tissues. Overall, compared with that of the type I genes, the expression of the type II genes was significantly higher. The CchMADS31 and CchMADS58 genes (type II) were highly expressed specifically in the flowers, which may in turn regulate the size of the flower meristem and petals. CchMADS55 was expressed specifically in the seeds, which might affect seed development. This study provides additional information for the functional characterization of the MADS-box gene family and lays an important foundation for in-depth study of related genes, such as those involved in the development of the reproductive organs of C. chekiangoleosa.

Effect of the PmARF6 Gene from Masson Pine (Pinus massoniana) on the Development of Arabidopsis.

Masson pine (Pinus massoniana) is a core industrial tree species that is used for afforestation in southern China. Previous studies have shown that Auxin Response Factors (ARFs) are involved in the growth and development of various species, but the function of ARFs in Masson pine is unclear. In this research, we cloned and identified Masson pine ARF6 cDNA (PmARF6). The results showed that PmARF6 encodes a protein of 681 amino acids that is highly expressed in female flowers. Subcellular analysis showed that the PmARF6 protein occurred predominantly in the nucleus and cytomembrane of Masson pine cells. Compared with wild-type (WT) Arabidopsis, transgenic Arabidopsis plants overexpressing PmARF6 had fewer rosette leaves, and their flower development was slower. These results suggest that overexpression of PmARF6 may inhibit the flower and leaf development of Masson pine and provide new insights into the underlying developmental mechanism.

The reference genome of camellia chekiangoleosa provides insights into camellia evolution and tea oil biosynthesis.

Camellia oil extracted from Camellia seeds is rich in unsaturated fatty acids (UFAs) and secondary metabolites beneficial to human health. However, no oil-tea tree genome has yet been published, which is a major obstacle to investigating the heredity improvement of oil-tea trees. Here, using both Illumina and PicBio sequencing technologies, we present the first chromosome-level genome sequence of the oil-tea tree species Camellia chekiangoleosa Hu. (CCH). The assembled genome consists of 15 pseudochromosomes with a genome size of 2.73 Gb and a scaffold N50 of 185.30 Mb. At least 2.16 Gb of the genome assembly consists of repetitive sequences, and the rest involves a high-confidence set of 64 608 protein-coding gene models. Comparative genomic analysis revealed that the CCH genome underwent a whole-genome duplication (WGD) event shared across the Camellia genus at ~57.48 MYA and a γ-WGT event shared across all core eudicot plants at ~120 MYA. Gene family clustering revealed that the genes involved in terpenoid biosynthesis have undergone rapid expansion. Furthermore, we determined the expression patterns of oleic acid accumulation- and terpenoid biosynthesis-associated genes in six tissues. We found that these genes tend to be highly expressed in leaves, pericarp tissues, roots, and seeds. The first chromosome-level genome of oil-tea trees will provide valuable resources for determining Camellia evolution and utilizing the germplasm of this taxon.

Integrative analysis of wood biomass and developing xylem transcriptome provide insights into mechanisms of lignin biosynthesis in wood formation of Pinus massoniana.

Lignin is an important renewable energy source as an excellent new battery fuel and ideal substitutes for the petrochemical industry. However, the molecular mechanism underlying lignin biosynthesis in wood formation of P. massoniana remains unexplored. Thus, an integrative analysis of wood biomass and the developing xylem transcriptome was performed to identify genes involved in lignin biosynthesis. A total of 1624 differentially expressed genes (DEGs) were identified, consisting of 797 upregulated and 827 downregulated genes (MaxG vs MinG). Additionally, 122 candidate genes and 17 DEGs were successfully annotated to the lignin biosynthesis pathway. All upregulated MYB and NAC genes were regulators of secondary cell wall formation. Moreover, the qRT-PCR analyses shown that 9 lignin biosynthesis-related genes and 7 transcription factor-encoding genes were upregulated (MaxG vs MinG), which indicated that the downregulation of lignin biosynthesis-related genes might be the possible causes of growth retardation and dwarf phenotype in some P. massoniana individuals. The identification of lignin biosynthesis-related genes can provide valuable genetic basis and resource for further researches on molecular mechanisms of lignin biosynthesis and contribute to the future investigations of bioengineering and synthetic biology to regulate lignin content in wood formation for the pulp and wood utilization industry.

Transcriptomic analysis of flower color variation in the ornamental crabapple (Malus spp.) half-sib family through Illumina and PacBio Sequel sequencing.

Ornamental crabapple is an important woody ornamental plant with flower colors ranging from white to pink to red, and the degree of redness is directly related to the anthocyanin content. To explore the molecular mechanism leading to the variation in flower color in ornamental crabapple, transcriptome sequencing using the Illumina and PacBio Sequel platforms revealed the difference in gene expression between the petals of plants with white and red flowers in the half-sib family. In total, the analysis identified 603 differentially expressed genes (DEGs), including 449 upregulated and 154 downregulated genes. GO and KEGG enrichment analyses of the DEGs showed that the oxidation-reduction process and catalytic activity were more active in red petals, and most of the DEGs were involved in secondary metabolite synthesis and plant hormone signaling. Among the 603 DEGs, 10 were enriched as structural genes. Transcription factors related to anthocyanin synthesis and five genes related to anthocyanin transport and degradation were highly expressed in red petals. In addition, this study found that five AUX gene signals were differentially expressed in the two petal types. The discovery of these DEGs indicates that plant endogenous hormones also exert a regulatory effect on flower color.

Comparison among three methods for obtaining chloroplast genome sequences from the conifer Pinus massoniana.

The chloroplast genome (CPG) is a powerful tool for phylogenetic studies. Many CPGs have been determined using NGS. However, the large nuclear-genome and difficult CPG-DNA separation in conifers limit their application in related research. In this study, three methods (PCR + Sanger, PCR + HiSeq, cpDNA+HiSeq) for obtaining the CPGs of Pinus massoniana were compared for sequence accuracy, time and cost. PCR + Sanger obtained the most accurate CPGs with advantages in cost (3.08$/kb) and time (2-3 days); PCR + HiSeq generated some DNA fragments with low depth, and the SNPs false-positive-rate (0.44) and sequencing error-rate (0.0265) of this method were higher than those of the cpDNA+HiSeq. Moreover, the cost (~6.17$/kb) and time (4-5 weeks) would significantly increase when HiSeq sequencing were outsourced to sequencing service company. Thus, for the study of intraspecific and interspecies variation in CPGs, CPG sequences can be obtained by comprehensive methods to bridge the method shortcomings. Scuh as sequence accuracy, cost and time.

Development and characterization of chloroplast microsatellite markers for Pinus massoniana and their application in Pinus (Pinaceae) species.

Pinus massoniana is one of the important afforestation and pioneer tree species, which is widely distribute in southern China. Chloroplast simple sequence repeat markers (cpSSRs) have been widely used in studies of tree genetics, phylogenetic and breeding. We sequenced the whole chloroplast genome sequences of P. massoniana using PCR and Sanger sequencing. A total of 71 cpSSRs were identified, among which mononucleotide repeats were predominant (70.42%). Seventeen primer pairs were developed and amplification tests were conducted with 15 P. massoniana individuals. Also, cross-species amplification tests were conducted among 15 individuals per Pinus species, including P. elliottii, P. bungeana, P. armandii, P. caribaea, P. tabulaeformis, P. taiwanensis and P. yunnanensis which revealed polymorphic information content ranging from 0.2 to 0.8 and average of haploid diversity (h) ranging from 0.29 to 0.63. In addition, the polymorphic cpSSRs were useful in distinguishing the sampled pine species, and could be powerful tool in phylogenetic studies.

Complete Chloroplast Genome of Pinus massoniana (Pinaceae): Gene Rearrangements, Loss of ndh Genes, and Short Inverted Repeats Contraction, Expansion.

The chloroplast genome (CPG) of Pinus massoniana belonging to the genus Pinus (Pinaceae), which is a primary source of turpentine, was sequenced and analyzed in terms of gene rearrangements, ndh genes loss, and the contraction and expansion of short inverted repeats (IRs). P. massoniana CPG has a typical quadripartite structure that includes large single copy (LSC) (65,563 bp), small single copy (SSC) (53,230 bp) and two IRs (IRa and IRb, 485 bp). The 108 unique genes were identified, including 73 protein-coding genes, 31 tRNAs, and 4 rRNAs. Most of the 81 simple sequence repeats (SSRs) identified in CPG were mononucleotides motifs of A/T types and located in non-coding regions. Comparisons with related species revealed an inversion (21,556 bp) in the LSC region; P. massoniana CPG lacks all 11 intact ndh genes (four ndh genes lost completely; the five remained truncated as pseudogenes; and the other two ndh genes remain as pseudogenes because of short insertions or deletions). A pair of short IRs was found instead of large IRs, and size variations among pine species were observed, which resulted from short insertions or deletions and non-synchronized variations between "IRa" and "IRb". The results of phylogenetic analyses based on whole CPG sequences of 16 conifers indicated that the whole CPG sequences could be used as a powerful tool in phylogenetic analyses.

Transcriptome sequencing and SNP detection in Phoebe chekiangensis.

BACKGROUND: Phoebe chekiangensis is a rare tree species that is only distributed in south-eastern China. Although this species is famous for its excellent wood properties, it has not been extensively studied at the molecular level. RESULTS: Here, the transcriptome of P. chekiangensis was sequenced using next-generation sequencing technology, and 75,647 transcripts with 48,011 unigenes were assembled and annotated. In addition, 162,938 putative single nucleotide polymorphisms (SNPs) were predicted and 25 were further validated using the Sanger method. CONCLUSION: The currently available SNP prediction software packages showed low levels of correspondence when compared. The transcriptome and SNPs will contribute to the exploration of P. chekiangensis genetic resources and the understanding of its molecular mechanisms.