Yuhan Sun

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.

Selection occurs within linear fruit and during the early stages of reproduction in Robinia pseudoacacia

BackgroundPollen donor compositions differ during the early stages of reproduction due to various selection mechanisms. In addition, ovules linearly ordered within a fruit have different probabilities of reaching maturity. Few attempts, however, have been made to directly examine the magnitude and timing of selection, as well as the mechanisms during early life stages and within fruit. Robinia pseudoacacia, which contains linear fruit and non-random ovule maturation and abortion patterns, has been used to study the viability of selection within fruit and during the early stages of reproduction. To examine changes in the pollen donor composition during the early stages of reproduction and of progeny originating from different positions within fruit, paternity analyses were performed for three early life stages (aborted seeds, mature seeds and seedlings) in the insect-pollinated tree R. pseudoacacia.ResultsSelection resulted in an overall decrease in the level of surviving selfed progeny at each life stage. The greatest change was observed between the aborted seed stage and mature seed stage, indicative of inbreeding depression (the reduced fitness of a given population that occurs when related individual breeding was responsible for early selection). A selective advantage was detected among paternal trees. Within fruits, the distal ends showed higher outcrossing rates than the basal ends, indicative of selection based on the order of seeds within the fruit.ConclusionsOur results suggest that selection exists both within linear fruit and during the early stages of reproduction, and that this selection can affect male reproductive success during the early life stages. This indicates that tree species with mixed-mating systems may have evolved pollen selection mechanisms to increase the fitness of progeny and adjust the population genetic composition. The early selection that we detected suggests that inbreeding depression caused the high abortion rate and low seed set in R. pseudoacacia.

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.

Development and Evaluation of a Novel Set of EST-SSR Markers Based on Transcriptome Sequences of Black Locust (Robinia pseudoacacia L.)

Black locust (Robinia pseudoacacia L. of the family Fabaceae) is an ecologically and economically important deciduous tree. However, few genomic resources are available for this forest species, and few effective expressed sequence tag-derived simple sequence repeat (EST-SSR) markers have been developed to date. In this study, paired-end sequencing was used to sequence transcriptomes of R. pseudoacacia by the Illumina HiSeq TM2000 platform, and EST-SSR loci were identified by de novo assembly. Furthermore, a total of 1697 primer pairs were successfully designed, from which 286 primers met the selection screening criteria; 94 pairs were randomly selected and tested for validation using polymerase chain reaction amplification. Forty-five primers were verified as polymorphic, with clear bands. The polymorphism information content values were 0.033–0.765, the number of alleles per locus ranged from 2 to 10, and the observed and expected heterozygosities were 0.000–0.931 and 0.035–0.810, respectively, indicating a high level of informativeness. Subsequently, 45 polymorphic EST-SSR loci were tested for amplification efficiency, using the verified primers, in an additional nine species of Leguminosae, 23 loci were amplified in more than three species, of which two loci were amplified successfully in all species. These EST-SSR markers provide a valuable tool for investigating the genetic diversity and population structure of R. pseudoacacia, constructing a DNA fingerprint database, performing quantitative trait locus mapping, and preserving genetic information.

Phytohormone and assimilate profiles in emasculated flowers of the black locust (Robinia pseudoacacia) during development

Emasculation and bagging of flowers, which are widely used in the controlled pollination of monoclinous plants, may induce premature senescence, flower abscission and low fruit set. To determine the mechanism responsible for these phenomena, levels of abscisic acid (ABA), jasmonic acid (JA), indole-3-acetic acid (IAA), ethylene, soluble sugars, reducing sugars and free amino acids in black locust (Robinia pseudoacacia) flowers subjected to different treatments were quantified at different developmental stages. The phytohormones and assimilates were also quantified in untreated flowers to investigate the presence of discernible patterns. The levels of ethylene and ABA in emasculated and bagged (EB) flowers increased prematurely compared with those of untreated flowers, whereas the content of reducing sugars in EB flowers decreased compared with that of untreated flowers. These results indicated that the premature increase in ethylene and ABA synthesis, and the decrease in reducing sugars content, in EB flowers may cause flower abscission and result in low fruit set, which may be relevant for assimilate applications and future research on the regulation of controlled pollinations with exogenous phytohormones.

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.

Evaluation of the Genetic Diversity and Differentiation of Black Locust (Robinia pseudoacacia L.) Based on Genomic and Expressed Sequence Tag-Simple Sequence Repeats

Understanding the genetic diversity and differentiation of the genetic resources of a species is important for the effective use and protection of forest tree resources. Ex situ development is a common method for the protection of genetic diversity and an essential resource for users who require ready access to a species’ germplasm. In this study, we collected seeds of black locust (Robinia pseudoacacia L.) from 19 provenances, covering most of its natural distribution; we randomly selected 367 tender leaves with well-grown and different maternal strains from this group for further analysis. Forty-eight simple sequence repeat (SSR) primers were successfully selected from 91 pairs of SSR primers using native-deformation polyacrylamide gel electrophoresis. In addition, we identified identical genotypes among all individuals and evaluated the quality of the markers. From this, 35 loci were confirmed for analyses of genetic diversity and differentiation of the black locust provenances, which contained 28 expressed sequence tag-derived simple sequence repeats (EST-SSRs) and 7 genomic DNA-derived simple sequence repeats (G-SSRs). We observed high genetic diversity among the native black locust provenances, from which Wright’s fixation index and molecular variance suggested that a majority of the genetic differentiation variation could be attributed to within-provenance differences. The genetic distance and identity results indicated that geographic distance was not a dominating factor influencing the distribution of black locust. This is the first study to evaluate provenance genetic variation in native black locust samples using two types of SSR markers, which provides a comprehensive theoretical basis for ex situ conservation and utilization of genetic resources, with an emphasis on breeding applications.

Ovule positions within linear fruit are correlated with nonrandom mating in Robinia pseudoacacia

Post-pollination processes can lead to nonrandom mating among compatible pollen donors. Moreover, morphological patterns of ovule development within linear fruits are reportedly nonrandom and depend on ovule position. However, little is known about the relationship between nonrandom mating and ovule position within linear fruit. Here, we combined controlled pollen competition experiments and paternity analyses on R. pseudoacacia to better understand nonrandom mating and its connection with ovule position. Molecular determination of siring success showed a significant departure from the expected ratio based on each kind of pollen mixture, suggesting a nonrandom mating. Outcrossed pollen grains, which were strongly favored, produced significantly more progeny than other pollen grains. Paternity analyses further revealed that the distribution of offspring produced by one specific pollen source was also nonrandom within linear fruit. The stylar end, which has a higher probability of maturation, produced a significantly higher number of outcrossed offspring than other offspring, suggesting a correlation between pollen source and ovule position. Our results suggested that a superior ovule position exists within the linear fruit in R. pseudoacacia, and the pollen that was strongly favored often preferentially occupies the ovules that were situated in a superior position, which ensured siring success and facilitated nonrandom mating.