Differences in Albizia odoratissima genetic diversity between Hainan Island and mainland populations in China.

Background: This study aimed at exploring unique population genetic characteristics of Albizia odoratissima (Linn. f) Benth on Hainan Island to provide a scientific basis for its rational utilization and protection. Methods: It analyzed the genetic diversity and structure of 280 individuals from 10 subpopulations of A. odoratissima from Hainan Island and Baise City using 16 expression sequence markers - simple sequence repeat markers. Results: The genetic diversity of Hainan population (I = 0.7290, He = 0.4483) was lower than that of the Baise population (I = 0.8722, He = 0.5121). Compared with the Baise population (Nm = 2.0709, FST = 0.1077), the Hainan Island population (Nm = 1.7519, FST = 0.1249) exhibited lower gene flow and higher degree of genetic differentiation. Molecular variance and genetic differentiation analyses showed that the main variation originated from individuals within the subpopulation. There were significant differences in the genetic structure between Hainan and Baise populations. It grouped according to geographical distance, consistent with the Mantel test results (R2 = 0.77, p = 0.001). In conclusion, the genetic diversity of the island A. odoratissima population was lower than that distributed on land, the two populations exhibited obvious genetic structure differences. Both the degrees of inbreeding and genetic differentiation were higher in the island population than in the land population.

SMRT and Illumina sequencing provide insights into mechanisms of lignin and terpenoids biosynthesis in Pinus massoniana Lamb.

Wood and oleoresin are important industrial raw materials with high economic value; however, their molecular formation and biosynthesis mechanisms in different tissues of Pinus massoniana remain unexplored. Therefore, we used single-molecule real-time sequencing technology (SMRT) and Illumina RNA sequencing to establish a transcriptome dataset and explore the expression pattern of genes related to secondary metabolites involved in wood formation and oleoresin biosynthesis in six different P. massoniana tissues. In total, 63.58 Gb of polymerase reads were obtained, including 41,407 isoforms with an average length of 1822 bp. We identified 3939 and 8785 isoforms and 161 and 481 transcription factors with tissue expression specificity and in the reproductive and vegetative organs, respectively. Eighty isoforms were annotated as cellulose synthases and 224 isoforms involved in lignin biosynthesis were enriched. Additionally, we identified 217 isoforms involved in the terpenoid biosynthesis pathway, with needles having the most tissue-specific genes for terpenoid biosynthesis. Some isoforms related to lignin biosynthesis were highly expressed in the xylem, according to the results of transcriptome sequencing and real-time quantitative reverse-transcription polymerase chain reaction. Our research confirmed the advantages of SMRT sequencing and provided valuable information for the transcriptional annotation of P. massoniana, which will be beneficial for producing better raw wood and oleoresin materials.

Protoplast isolation and transcriptome analysis of developing xylem in Pinus massoniana (Pinaceae).

BACKGROUND: With active physiological and biochemical activities, tissue-specific protoplasts from cambial derivatives, could serve as a specific source for information on xylogenesis for softwood species resistant to stable genetic transformation and lacking available mutants. METHODS AND RESULTS: In this study, protoplasts were isolated from developing xylem of the Chinese red pine, Pinus massoniana, by enzymolysis. High-quality RNAs were extracted from developing xylem and their protoplasts for constructing transcriptome libraries. Using Illumina HiSeq 2500 PE150 platform, a total of 362,328,426 clean paired-end reads (54.35G) were generated from multiple cDNA libraries and assembled into 146,422 unigenes. The transcriptome data were further analysed to identify 1567 differentially expressed genes (DEGs) between the isolated protoplasts and developing xylem of P. massoniana (Masson pine), 1126 DEGs were upregulated in protoplasts relative to developing xylem cells and 441 were downregulated. Most of the differentially expressed genes in biological process terms are related to plant response, which may be due to the response to cell wall removal. Further, the expression pattern of 71 unigenes involved in lignin biosynthesis was verified by RNA-seq. CONCLUSIONS: This study is the first to report the transcriptome profiles of the developing xylem and its protoplasts of coniferous trees, which provide a new perspective and valuable resource for tracking transcriptional regulatory events in wood formation of Masson pine.

Uncovering miRNA-mRNA Regulatory Modules in Developing Xylem of Pinus massoniana via Small RNA and Degradome Sequencing.

Xylem is required for the growth and development of higher plants to provide water and mineral elements. The thickening of the xylem secondary cell wall (SCW) not only improves plant survival, but also provides raw materials for industrial production. Numerous studies have found that transcription factors and non-coding RNAs regulate the process of SCW thickening. Pinus massoniana is an important woody tree species in China and is widely used to produce materials for construction, furniture, and packaging. However, the target genes of microRNAs (miRNAs) in the developing xylem of P. massoniana are not known. In this study, a total of 25 conserved miRNAs and 173 novel miRNAs were identified via small RNA sequencing, and 58 differentially expressed miRNAs were identified between the developing xylem (PM_X) and protoplasts isolated from the developing xylem (PM_XP); 26 of these miRNAs were significantly up-regulated in PM_XP compared with PM_X, and 32 were significantly down-regulated. A total of 153 target genes of 20 conserved miRNAs and 712 target genes of 113 novel miRNAs were verified by degradome sequencing. There may be conserved miRNA-mRNA modules (miRNA-MYB, miRNA-ARF, and miRNA-LAC) involved in softwood and hardwood formation. The results of qRT-PCR-based parallel validation were in relatively high agreement. This study explored the potential regulatory network of miRNAs in the developing xylem of P. massoniana and provides new insights into wood formation in coniferous species.

High-throughput sequencing-based assembly of chloroplast genomes of five pine tree species.

Pinus plants are the largest existing group of gymnosperms and one of the most highly differentiated taxa. Due to its huge ecological, economic, and scientific value, the genetic diversity and the relationship between the intraspecific evolution of Pinus plants have gained wide attention. In this study, the chloroplast genomes of several common pine trees in southwest and south China, including P. massoniana (masson pine), P. yunnanensis (yunnan pine), P. latteri (south asia pine), P. crassicorticea (la ya pine), and P. elliottii (slash pine), and entire cpDNA sequences were obtained. Characteristics including the structure, repeated sequence, and codon bias of the cpDNA for these five pine tree species were analyzed.

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.