Inbreeding in Chinese Fir: Insight into the Rare Self-Fertilizing Event from a Genetic View.

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).

Further Mining and Characterization of miRNA Resource in Chinese Fir (Cunninghamia lanceolata).

In this study, we aimed to expand the current miRNA data bank of Chinese fir (Cunninghamia lanceolata (Lamb.) Hook.) regarding its potential value for further genetic and genomic use in this species. High-throughput small RNA sequencing successfully captured 140 miRNAs from a Chinese fir selfing family harboring vigor and depressed progeny. Strikingly, 75.7% (n = 106) of these miRNAs have not been documented previously, and most (n = 105) of them belong to the novel set with 6858 putative target genes. The new datasets were then integrated with the previous information to gain insight into miRNA genetic architecture in Chinese fir. Collectively, a relatively high proportion (62%, n = 110) of novel miRNAs were found. Furthermore, we identified one MIR536 family that has not been previously documented in this species and four overlapped miRNA families (MIR159, MIR164, MIR171_1, and MIR396) from new datasets. Regarding the stability, we calculated the secondary structure free energy and found a relatively low R2 value (R2 < 0.22) between low minimal folding free energy (MFE) of pre-miRNAs and MFE of its corresponding mature miRNAs in most datasets. When in view of the conservation aspect, the phylogenetic trees showed that MIR536 and MIR159 sequences were highly conserved in gymnosperms.

Genetic Characterization of Chinese fir from Six Provinces in Southern China and Construction of a Core Collection.

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.

Somatic Embryogenesis of Immature Cunninghamia lanceolata (Lamb.) Hook Zygotic Embryos.

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.

The complete chloroplast genome sequence of Cunninghamia lanceolata.

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.

Global Reprogramming of Transcription in Chinese Fir (Cunninghamia lanceolata) during Progressive Drought Stress and after Rewatering.

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.