Tree peony (Paeonia suffruticosa Andrews) is a perennial woody shrub bearing large and colorful flowers. However, the flowering period is short and relatively uniform, which to an important extent hinders the cultivation and exploitation of ornamental peonies. In this study, the segregation of an F₁ population derived from P. ostti ‘Feng Dan’ (an early-flowering cultivar) × P. suffruticosa ‘Xin Riyuejin’ (a late-flowering cultivar) was used to screen and analyze candidate genes associated with flowering period of the two parents. Extreme early- and late-flowering genotypes of the F₁ population at full-bloom stage were sampled to establish an early-flowering mixed pool (T03), a late-flowering mixed pool (T04), a late-flowering male pool (T01), and an early-flowering female pool (T02), using the Sequencing By Synthesis (SBS) technology on the Illumina HiSeq TM2500 platform. A total of 56.51 Gb of clean reads data, comprising at least 87.62% of Quality30 (Q30), was generated, which was then combined into 173,960 transcripts (N50 = 1781) and 78,645 (N50 = 1282) unigenes, with a mean length of 1106.76 and 732.27 base pairs (bp), respectively. Altogether, 58,084 genes were annotated by comparison with public databases, based on an E-value parameter of less than 10⁻⁵ and 10⁻¹⁰ for BLAST and HMMER, respectively. In total, 291 unigene sequences were finally screened out by BSR-seq (bulked segregant RNA-seq) association analysis. To validate these unigenes, we finally confirmed seven unigenes that were related to early and late flowering, which were then verified by quantitative real-time PCR (qRT-PCR). This is the first reported study to screen genes associated with early and late flowering of tree peony by the BSA (bulked sample analysis) method of transcriptome sequencing, and to construct a high-quality transcriptome database. A set of candidate functional genes related to flowering time was successfully obtained, providing an important genetic resource for further studies of flowering in peony and the mechanism of regulation of flowering time in tree peony.

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.

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.

Prunella vulgaris, an essential component of traditional Chinese medicine, is suitable for growing in soil with a pH value ranging from 6.5 to 7.5. However, it is primarily cultivated in acidic soil regions of China, where its growth is frequently compromised by acidic stress. Selenium (Se) has been recognized for its potential to enhance stress tolerance in plants. However, its role in acid-stress-induced oxidative stress is not clear. In this study, the effects of varying Se concentrations on the growth and quality of P. vulgaris under acidic stress were investigated. The results showed that acid stress enhanced antioxidant enzyme activities, non-enzymatic antioxidant substances, and osmolyte content, accompanied by an increase in oxidant production and membrane damage. Furthermore, it decreased the photosynthetic capacity, inhibited root and shoot growth, and diminished the yield of P. vulgaris. In contrast, exogenous application of Se, particularly at 5 mg L⁻¹, markedly ameliorated these adverse effects. Compared to acid-stressed plants, 5 mg L⁻¹ Se treatment enhanced superoxide dismutase, peroxidase, ascorbate peroxidase, and glutathione peroxidase activities by 150.19%, 54.94%, 43.43%, and 45.55%, respectively. Additionally, soluble protein, soluble sugar, and proline contents increased by 11.75%, 23.32%, and 40.39%, respectively. Se application also improved root architecture and alleviated membrane damage by reducing hydrogen peroxide, superoxide anion, malondialdehyde, and electrolyte leakage levels. Furthermore, it significantly enhanced the photosynthetic capacity by elevating pigment levels, the performance of PSI and PSII, electron transfer, and the coordination of PSI and PSII. Consequently, plant growth and spica weight were significantly promoted, with a 12.50% increase in yield. Moreover, Se application upregulated key genes involved in flavonoid and phenolic acid metabolic pathways, leading to elevated levels of total flavonoids, caffeic acid, ferulic acid, rosmarinic acid, and hyperoside by 31.03%, 22.37%, 40.78%, 15.11%, and 20.84%, respectively, compared to acid-stressed plants. In conclusion, exogenous Se effectively alleviated the adverse effects of acid stress by improving the antioxidant system, growth, and photosynthetic capacity under acid stress, thus enhancing the yield and quality of P. vulgaris.

The DREB transcription factors regulate multiple stress response genes, and are therefore useful for molecular plant breeding. AhDREB, a stress-inducible gene, was isolated from Atriplex hortensis L. and introduced into Populus tomentosa Carrière under the control of the CaMV35S promoter. Under salt stress, the chlorophyll content and net photosynthetic rate were higher in transgenic lines than in the wild type (WT). Moreover, the rate of electrolyte penetration (REC) was lower in the transgenic lines. Additional analyses revealed that the AhDREB transgenic plants generally displayed lower malondialdehyde (MDA) activity but higher superoxide dismutase (SOD) and peroxidase (POD) activities and proline content than the WT under salt stress. RNA sequencing indicated that AhDREB could enhance tolerance to salt by activating various downstream genes in the transgenic plants. Furthermore, no growth inhibition was detected in transgenic plants expressing AhDREB driven by the constitutive CaMV35S promoter. The transcriptome showed 165 and 52 differentially expressed genes in transgenic plants under stress and non-stress conditions, respectively, among which no significant metabolic pathway was enriched and no unintended effects have yet been identified. Together, these results suggest that AhDREB may be a good candidate gene for increasing salt tolerance in transgenic poplar breeding.

Populus tomentosa grow rapidly, but are salt susceptible. To quickly and efficiently gain new poplar breeds with better salt resistance, a DREB transcription factor derived from Atriplex hortensis was transformed into triploid Populus tomentosa by our lab, which significantly improved the salt tolerance of host plants. However, environmental impacts of transgenic plants must be assessed before large-scale cultivation in China. Here, we conducted a field trial of AhDREB1 transgenic and non-transgenic triploid Populus tomentosa to assess the impact of transgenic trees on rhizospheric soil microbial communities and allelopathic activity of leaves. No significant differences in the number of soil microbes present were detected between the transgenic lines and the non-transgenic controls. The allelopathic activity of leaves from both the transgenic and non-transgenic lines varied with sampling time, but did not differ significantly between the transgenic and non-transgenic lines. These results indicate that the impact on the environment of AhDREB1 transgenic P. tomentosa did not differ significantly from that of the non-transformed controls for the variables observed in this field trial. We also investigated the persistence of AhDREB1 genes in decomposing transgenic poplar leaf on the soil under natural conditions for five months, and our data indicated that fragments of the genetically modified DNA were not detectable in the field after more than two months. We used a triphenyl tetrazolium chloride test (TTC) (or pollen germination method) and hybridization to test the pollen viability and fertility, respectively, of the transgenic and non-transgenic trees and the results showed that the pollen viability of both the transgenic and non-transgenic trees was extremely low in 2016; the receptor plant may have been sterile.