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.

Genome-Wide Association Study With Growth-Related Traits and Secondary Metabolite Contents in Red- and White-Heart Chinese Fir.

Chinese fir [Cunninghamia lanceolata (Lamb.) Hook] is an important evergreen coniferous tree species that is widely distributed in many southern provinces of China and has important economic value. The Chinese fir accounts for 1/4 and 1/3 of the total artificial forest area and stock volume, respectively. Red-heart Chinese fir is popular in the market because of its high density and red heartwood. The long-growth cycle hindered the breeding process of Chinese fir, while molecular marker-assisted breeding could accelerate it. However, Chinese fir, a perennial conifer species, has a large genome, which has not yet been published. In this study, the growth-related traits and secondary metabolite contents of red- and white-heart Chinese fir were measured and found to be different between them. There are extremely significant differences among growth-related traits (p < 0.001), but secondary metabolite contents have different correlations due to differences in chemical structure. Moreover, genotype effect analysis of the substantially correlated single nucleotide polymorphisms (SNPs) revealed that most of the loci related to each growth-related traits were different from each other, indicating a type specificity of the genes regulated different growth-related traits. Furthermore, among the loci related to secondary metabolite contents, nine loci associated with multiple metabolite phenotypes such as Marker21022_4, Marker21022_172, Marker24559_31, Marker27425_37, Marker20748_85, Marker18841_115, Marker18841_198, Marker65846_146, and Marker21486_163, suggesting the presence of pleiotropic genes. This study identified the potential SNP markers associated with secondary metabolites in Chinese fir, thus setting the basis for molecular marker-assisted selection.

Characterization of the Transcriptome and Gene Expression of Tetraploid Black Locust Cuttings in Response to Etiolation.

Etiolation (a process of growing plants in partial or complete absence of light) promotes adventitious root formation in tetraploid black locust (Robinia pseudoacacia L.) cuttings. We investigated the mechanism underlying how etiolation treatment promotes adventitious root formation in tetraploid black locust and assessed global transcriptional changes after etiolation treatment. Solexa paired-end sequencing of complementary DNAs (cDNAs) from control (non-etiolated, NE) and etiolated (E) samples resulted in 107,564 unigenes. In total, 52,590 transcripts were annotated and 474 transcripts (211 upregulated and 263 downregulated) potentially involved in etiolation were differentially regulated. These genes were associated with hormone metabolism and response, photosynthesis, signaling pathways, and starch and sucrose metabolism. In addition, we also found significant differences of phytohormone contents, activity of following enzymes i.e., peroxidase, polyphenol oxidase and indole acetic acid oxidase between NE and E tissues during some cottage periods. The genes responsive to etiolation stimulus identified in this study will provide the base for further understanding how etiolation triggers adventitious roots formation in tetraploid black locus.

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.

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.