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Cunninghamia lanceolate (Lamb.) Hook. (Chinese fir) is one of the most important wood-producing species, supplying ~20% of commercial timber by plantations in China. However, the genetic potential of the bred variety is limited by soil degrading in the long term and requiring continuous replanting, and especially the shortage and supply of active and efficient phosphorus. Recently, great attention has been paid to the genotypic variation in phosphorus conversion and utilization efficiency by tree breeders. In this study, the morphological characteristics were used to evaluate the Chinese fir clonal Pi-efficiency stress. A Pi-tolerant clone and a Pi-sensitive clone were selected for RNA sequencing, respectively. In addition, gene function annotation and weighted correlation network analysis (WGCNA) were performed. A total of 60 hub genes were selected, combining phosphate accumulation under Pi-deficiency stress. We also used RNA-seq data to analyze the differences in the response of Pi-sensitive clones and Pi-tolerant clones to Pi-deficiency stress, and real-time quantitative polymerase chain reaction (RT-PCR) analyses were used to test the validity of transcriptome data. The present study provided new insights into the molecular mechanisms of Pi-efficient utilization in Chinese fir clones.

Chinese fir (Cunninghamia lanceolata (Lamb.) Hook.) is a major timber conifer species in southern China. In this study, we aimed to capture the rarely advanced phenomenon for selfing in this species and illustrated the underlying molecular mechanism, especially the hub gene-regulated networks and path­ways, by global transcriptome analysis assays (RNA-Seq). Self-pollination trials revealed a wide variation of selfing effects among parents. Parent cx569 produced a selfed family with the best growth performance at the seedling stage. The growth-based extremely advanced (AD) (n=3) and depressed (DE) variants (n=3; different types) were then subjected to comparative RNA-Seq. The transcriptome data revealed more than 5000 differentially expressed genes (DEGs) for each comparison group (AD versus DE). Weighted gene co-expression network analysis (WGCNA) further identified more than 80 important DEGs that were significantly associated with growth traits in each comparison group. A subsequent enrichment analy­sis showed that the identified DEGs belonged to six main types, including xylem metabolism-related, sugar and energy metabolism-related, plant hormone signal transduction-related, stress response-related, cytochrome-related, and transcription factor genes. Ten hub genes represented by the ERF071, MYB-relate 305, WRKY6, WRKY31, PER3, LAC4, CESA8, CESA9, GID1, and PR1 genes were co-identified between AD and DE variants. These genes exhibited rather different expression patterns between AD and DE variants, especially of the transcription factor ERF071 gene that presented a low transcript level in the AD seedlings with only 4.45% activity compared to DE's. While, the plant hormone signal transduction GID1 gene was significantly upregulated in AD by about 20-fold when compared to DE's, and fold change of the lignin biosynthesis-related PER3, CESA9 and LAC4 gene expression parallel reached to 10–15 times in an upreg­ulation pattern in AD seedlings. The set of hub gene-linked interaction networks and pathways revealed in this study may be responsible for the rarely advanced phenomenon for selfing at the seedling stage in Chinese fir.

In this study, we aimed to expand the current miRNA data bank of Chinese fir (

Chinese fir (Cunninghamia lanceolata (Lamb.) Hook.) is a fast-growing conifer with great forestation value and prefers outcrossing with high inbreeding depression effect. Previously, we captured a special Chinese fir parent clone named as ‘cx569’ that lacks early inbreeding depression. In view of the fact that very little has been published about the rare self-fertilizing event in Chinese fir from a genetic view, herein, we conduct an SSR-based study on the variation of open- and self-pollinated offspring of this parent to gain a view of the rare self-fertilizing event. The results indicated that genetic diversity of self-pollinated offspring was significantly reduced by half (Ho: 0.302, vs. 0.595, p = 0.001; He: 0.274 vs. 0.512, p = 0.002) when compared to an open-pollinated set. Self-pollinated offspring also had significantly positive FIS values (FIS = 0.057, p = 0.034) with a much higher proportion of common allele (20.59% vs. 0), reflecting their heterozygote deficiency. Clustering analysis further indicated a separation of the self- and opened- pollinated groups, implying a natural preference of outcrossing for cx569. However, the cx569 still had 6% acceptance for selfing. When accepted 100% for its own pollen, the cx569 led to a genetically unique selfing group. Additionally, this selfing group seemed to be consistently homozygous at seven particular loci. These findings gave us more genetic clues to gain insight into the rare self-fertilizing event in conifer (Chinese fir).

Accurate estimation of genetic relationships among breeding materials and their genetic diversity contributes to the optimal design of breeding programs. For Chinese fir (Cunninghamia lanceolata (Lamb.) Hook.), an important indigenous tree species in China, breeders have attempted to employ different molecular markers to address the genetic architecture of their target population, but the power of an advanced parent tree population with a complex pedigree relationship is still rather limited. In this study, a partly known pedigree map combined with marker-derived (SSRs and SNPs) information was implemented for the first time in the assessment of the genetic relatedness of a complex advanced parent tree population (n = 50) in Chinese fir. The bivariate analysis showed that relatedness coefficients between individuals based on SSRs were significantly correlated with SNPs (r = 0.690, p < 0.01). Moreover, the heatmap generated by the SSR-based coefficient matrix was largely consistent with that derived from the SNP-based matrix. Additionally, STRUCTURE and ADMIXTURE analyses based on the two markers showed an analogical genetic clustering result. When compared to the recorded pedigree information, the genetic relationships estimated by the two molecular markers were broadly parallel with pedigree relatedness. These results indicated that SSRs and SNPs can be used as effective tools to clarify genetic relationships when complete pedigree records are not available in Chinese fir. Based on the two markers, the present study revealed a relatively wide genetic variation (SSRs: PIC = 0.573; SNPs: PIC = 0.231) in the selected parent trees. This investigation provides important input into the progress of Chinese fir advanced-generation breeding.

Large ex situ germplasm collections of plants generally contain significant diversity. A set of 700 well-conserved Chinese fir (Cunninghamia lanceolata (Lamb.) Hook) clones from six provinces in southern China in the ex situ gene bank of Longshan State Forest, was analyzed using 21 simple sequence repeat markers, with the aim of assessing the genetic diversity of these germplasm resources. Genetic analysis revealed extensive genetic variation among the accessions, with an average of 8.31 alleles per locus and a mean Shannon index of 1.331. Excluding loci with null alleles, we obtained a low level of genetic differentiation among provinces, consistent with the interpopulation genetic variation (1%). Three clusters were identified by STRUCTURE, which did not match the individuals’ geographical provenances. Ten traits related to growth and wood properties were quantified in these individuals, and there was substantial variation in all traits across individuals, these provide a potential source of variation for genetic improvement of the Chinese fir. Screening large collections for multiple-trait selective breeding programs is laborious and expensive; a core collection of 300 accessions, representative of the germplasm, was established, based on genotypic and phenotypic data. The identified small, but diverse, collections will be useful for further genome-wide association studies.

Two efficient somatic embryogenesis systems were developed in Chinese fir, the most important conifer for industrial wood production in China. Three development stages (cleavage polyembryony, dominant embryo, and precotyledon) of immature embryos derived from 25 genotypes of open-pollinated mother trees were used as initial explants. Cleavage polyembryony-stage embryos with a 12.44% induction rate was the most embryogenic response stage. The highest frequency of embryogenic callus (13.86%) induction was obtained from DCR medium supplemented with 1.5 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.3 mg L−1 kinetin (KN). An average of 53.33 early somatic embryos were produced from approximately 0.2 g (fresh weight) embryogenic callus after 2 weeks of incubation on medium supplemented with 50 μmol L−1 abscisic acid (ABA) and 100 g L−1 polyethylene glycol (PEG) 6000. About 53% dominant embryos have an embryogenic response after a 6-week cultivation on medium supplemented with 1.0–2.0 mg L−1 benzyladenine (BA), 0.2 mg L−1 naphthylacetic acid (NAA) or 2,4-D, and 0.004 mg L−1 thidiazuron (TDZ). After three successive transfer cultures on medium supplemented with 1.5 mg L−1 BA, 0.2 mg L−1 NAA, and 0.004 mg L−1 TDZ, 4.49–16.51% of the embryos developed into somatic embryos.

Chinese fir [Cunninghamia lanceolata (Lamb.) Hook] is a major timber tree species in China. In the present study, sequence-related amplified polymorphism (SRAP) markers were used to evaluate the polymorphisms, diversity, and relationships in a collection of Chinese fir elite genotypes (n = 103) from the Lechang provenance, an endangered provenance. Thirty-five SRAP primer combinations produced 620 bands among the tested 103 Lechang provenance genotypes and 17 non-Lechang provenance genotypes; 577 (93.1 %) of these were polymorphic, yielding an average of 16.5 polymorphic bands per primer combination. Of the SRAP primer combinations (n = 35), 14 ones identified ≥70.0 % of the 120 genotypes. The Me12–Em19 combination had the strongest discriminatory ability with 93.3 % of the genotypes identified. An UPGMA dendrogram further showed that most of the Lechang provenance genotypes (87.4 %) grouped together (Cluster A). The Lechang genotypes had a considerable amount of genetic variation with genetic similarity from 0.40 to 0.81. The current work may facilitate the management of the endangered Lechang Chinese fir provenance.

A provenance trial of Michelia chapensis Dandy in the south of China was carried out in three sites to assess yield and stability analyses. The trials were conducted as randomized, complete block designs with four replications at each site. Additive main effects and multiplicative interaction (AMMI) and genotype main effect and genotype × environment interaction (GGE) were employed in the evaluation of provenances; AMMI analyses showed that significant genotype × environment interaction effects (P

Chinese fir (Cunninghamia lanceolata), an evergreen conifer, is the most commonly grown afforestation species in southeast China due to its rapid growth and good wood qualities. To gain a better understanding of the drought-signalling pathway and the molecular metabolic reactions involved in the drought response, we performed a genome-wide transcription analysis using RNA sequence data. In this study, Chinese fir plantlets were subjected to progressively prolonged drought stress, up to 15 d, followed by rewatering under controlled environmental conditions. Based on observed morphological changes, plantlets experienced mild, moderate, or severe water stress before rehydration. Transcriptome analysis of plantlets, representing control and mild, moderate, and severe drought-stress treatments, and the rewatered plantlets, identified several thousand genes whose expression was altered in response to drought stress. Many genes whose expression was tightly coupled to the levels of drought stress were identified, suggesting involvement in Chinese fir drought adaptation responses. These genes were associated with transcription factors, signal transport, stress kinases, phytohormone signalling, and defence/stress response. The present study provides the most comprehensive transcriptome resource and the first dynamic transcriptome profiles of Chinese fir under drought stress. The drought-responsive genes identified in this study could provide further information for understanding the mechanisms of drought tolerance in Chinese fir.

Knowledge on population diversity and structure is of fundamental importance for conifer breeding programs. In this study, we concentrated on the development and application of high-density single nucleotide polymorphism (SNP) markers through a high-throughput sequencing technique termed as specific-locus amplified fragment sequencing (SLAF-seq) for the economically important conifer tree species, Chinese fir (Cunninghamia lanceolata). Based on the SLAF-seq, we successfully established a high-density SNP panel consisting of 108,753 genomic SNPs from Chinese fir. This SNP panel facilitated us in gaining insight into the genetic base of the Chinese fir advance breeding population with 221 genotypes for its genetic variation, relationship and diversity, and population structure status. Overall, the present population appears to have considerable genetic variability. Most (94.15%) of the variability was attributed to the genetic differentiation of genotypes, very limited (5.85%) variation occurred on the population (sub-origin set) level. Correspondingly, low FST (0.0285&ndash, 0.0990) values were seen for the sub-origin sets. When viewing the genetic structure of the population regardless of its sub-origin set feature, the present SNP data opened a new population picture where the advanced Chinese fir breeding population could be divided into four genetic sets, as evidenced by phylogenetic tree and population structure analysis results, albeit some difference in membership of the corresponding set (cluster vs. group). It also suggested that all the genetic sets were admixed clades revealing a complex relationship of the genotypes of this population. With a step wise pruning procedure, we captured a core collection (core 0.650) harboring 143 genotypes that maintains all the allele, diversity, and specific genetic structure of the whole population. This generalist core is valuable for the Chinese fir advanced breeding program and further genetic/genomic studies.

To determine the phenotypic variation in 700 ten-year grafted Chinese fir collected from six provinces in southern China, 10 phenotypic traits were investigated: tree height, diameter at breast height, bark thickness, volume of timber, heartwood ratio, density of wood, hygroscopicity, tracheid length, tracheid diameter, and ratio of tracheid length to tracheid diameter. Abundant phenotypic variation was found among the six populations; the phenotypic variation coefficients all exceeded 10%, and the largest was for volume of timber. Significant variation (p < 0.01 or 0.05) in traits was found among the populations, except for diameter at breast height, heartwood ratio, and tracheid diameter, while all traits differed significantly (p < 0.01) within populations. The high value of repeatability (broad-sense heritability) suggested moderate genetic control of the traits. The 10 traits were strongly correlated within the entire population; strong positive correlations (p < 0.01) were observed between growth traits, and significant negative correlations (p < 0.01 or 0.05) were found between the density of wood and most other characteristics, except for heartwood ratio and ratio of tracheid length to tracheid diameter. Using diameter at breast height and density of wood as criteria, 98 relatively fast-growing genotypes with relatively high wood basic density were identified.

We determined the complete chloroplast genome sequence of Cunninghamia lanceolata (GenBank accession: NC_021437.1) in this study. The total length of the chloroplast genome is 135 334 bp. The GC content is 35%. A total of 119 genes are successfully annotated, including 35 tRNA (20 tRNA species), 3 rRNA (3 rRNA species) and 81 protein-coding genes (81 PCG species). Twelve protein-coding genes (rps16, ycf3, rpoC1, atpF, rps12, ndhB, rpl2, rpl16, petD, petB, ndhA, rps15) contain one or two introns. A maximum likelihood phylogenetic analysis showed that this newly characterized Cunninghamia lanceolata chloroplast genome will provide essential data for further study on phylogenetic resolution, biodiversity for the genus Cunninghamia and Taxodiacea.