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Drought stress in trees limits their growth, survival, and productivity and it negatively affects the afforestation survival rate. Our study focused on the molecular responses to drought stress in a coniferous species Larix olgensis A. Henry. Drought stress was simulated in one-year-old seedlings using 25% polyethylene glycol 6000. The drought stress response in these seedlings was assessed by analyzing select biochemical parameters, along with gene expression and metabolite profiles. The soluble protein content, peroxidase activity, and malondialdehyde content of L . olgensis were significantly changed during drought stress. Quantitative gene expression analysis identified a total of 8172 differentially expressed genes in seedlings processed after 24 h, 48 h, and 96 h of drought stress treatment. Compared with the gene expression profile of the untreated control, the number of up-regulated genes was higher than that of down-regulated genes, indicating that L . olgensis mainly responded to drought stress through positive regulation. Metabolite analysis of the control and stress-treated samples showed that under drought stress, the increased abundance of linoleic acid was the highest among up-regulated metabolites, which also included some saccharides. A combined analysis of the transcriptome and metabolome revealed that genes dominating the differential expression profile were involved in glutathione metabolism, galactose metabolism, and starch and sucrose metabolism. Moreover, the relative abundance of specific metabolites of these pathways was also altered. Thus, our results indicated that L . olgensis prevented free radical-induced damage through glutathione metabolism and responded to drought through sugar accumulation.

In forest genetics research, precise evaluation of half-sib families provides essential insights for the selection and improvement of key species. This study systematically examined 40 half-sib families of L. olgensis from northeast China, analyzed 21 traits related to growth, form, wood, photosynthesis, and physiological traits. The research employed analysis of variance (ANOVA), genetic parameter estimation, and correlation analysis to assess family variation and trait relationships. The results indicated that 16 traits differed significant or highly significant ( P  < 0.05) among families. The coefficient of variation (CV) ranged from 7.78% to 65.16%, and family heritability ranged from 0.037 to 0.835. Wood traits showed negative correlations with growth and form traits. Based on average realized gains, we identified the estimation method of breeding value as optimal, leading to the selection of eight superior families at a 20% selection rate, with genetic gains ranged from 1.98% to 65.55%. The realized gains for tree height, diameter at breast height, volume, crown width, straightness, branching angle, lateral branch thickness, wood density, hemicellulose, holocellulose, and lignin were 5.97%, 8.11%, 20.44%, 10.32%, 3.06%, 3.22%, 10.74%, 1.99%, – 1.26%, – 1.36%, and 2.57%, respectively. These findings demonstrate that multi-trait, breeding-value-based selection effectively improves L. olgensis . This study provides both a theoretical basis and practical guidance for the genetic improvement of this economically important species.

Background Larch ( Larix gmelinii (Rupr.) Kuzen.) is an important timber and ecological tree species in northern China. Excellent germplasm resources have been acquired through time-consuming traditional breeding. Molecular breeding offers a promising approach to shorten the breeding cycle and achieve genetic improvements more efficiently. MicroRNAs (miRNAs) are non-coding, single-stranded small RNAs that primarily affect plant growth and stress resistance, including drought stress. However, the study of miRNAs in larch under drought stress has not been well explored. Results In this study, to investigate the function of Lol-miR11467 under PEG osmotic stress in larch, embryogenic callus tissue of Larix kaempferi 3 × L. gmelinii 9 was employed as the experimental material, serving as the explants for this study. Lol-miR11467 was transferred into the explants using an Agrobacterium-mediated method to determine the physiological changes and survey gene expression changes in overexpressing Lol-miR11467 cell lines. The results showed that the fresh weight, peroxidase (POD), soluble protein and soluble sugar content of the overexpressing Lol-miR11467 were lower than that of the wild-type, while malondialdehyde (MDA) content increased under PEG osmotic stress. Transcriptome analysis showed that genes associated with phenylpropanoid metabolism, transcription factors, oxidoreductase, plant hormone signal transduction, glucose metabolism and bioprotective macromolecules were mainly downregulated in Lol-miR11467 cell lines. Conclusions Overall, these results indicated that the drought resistance of the overexpressing Lol-miR11467 cell lines was reduced. This study’s findings might provide a foundation for understanding the molecular mechanisms of miRNAs under PEG osmotic stress in larch, potentially contributing to the development of strategies for improving plant resilience to environmental stresses.

Korean pine is a native tree species in Northeast China. In order to meet the needs of germplasm resource evaluation and molecular marker-assisted breeding of Korean pine, we collected Korean pine clones from 7 populations in Northeast China, analyzed the genetic diversity and genetic structure by SSR molecular marker technology and clustered them to revealed the inter- and intrapopulation differentiation characteristics of each clone. The fingerprint profiles of 161 Korean pine clones were also constructed. 77 alleles were detected for 11 markers, and 18 genotypes were identified on average for each marker. The PIC of the different markers ranged from 0.155-0.855, and the combination of PI and PIsibs for the 11 markers was 3.1 × 10 -8 and 1.14 × 10 -3 , respectively. MANOVA showed that genetic variation existed mainly within populations, accounting for 98% of the total variation. The level of genetic differentiation among populations was low, with an average Nm between populations of 11.036. Genetic diversity is lower in the Lushuihe population and higher in the Tieli population. The 161 Korean pine clones were divided into 4 or 7 populations, and the 7 populations were not clearly distinguished from each other, with only the Lushuihe population showing partial differentiation. There is no significant correlation between the genetic distance of Korean pine populations and the geographical distance of their superior tree sources. This result can provide recommendations for future Korean pine breeding programs. The combination of 11 markers could completely distinguish 161 clones and establish the fingerprint. Genetic diversity of Korean pine clones from the 7 populations was abundant, and the genetic distances of individuals and populations were evenly dispersed. The fingerprint map can be used for the identification of Korean pine clones.

Background The principal objective of this research is to develop highly specific polymorphic molecular markers, with the aim of addressing the paucity of simple sequence repeat (SSR) markers in Pinus koraiensis . The objective of this initiative is to facilitate the efficient management of genetic resources within Pinus koraiensis , enable precise pedigree identification and establish a foundational framework for subsequent whole-genome sequencing and assembly strategies. To achieve this objective, a simplified genome sequencing approach was employed, utiliZing RAD-Seq technology on a sample of 100 clones of Pinus koraiensis sourced from 6 seed orchards. The SSR sequences present within the contig were identified via TBtools-II software, which was also employed to generate a comprehensive summary and analysis of the sequence characteristics. On the Basis of these insights, primers were designed and subjected to meticulous screening using bioinformatics methodologies, and their efficacy was subsequently verified. The SSR markers were subsequently employed to examine the genetic diversity and structure of the plus tree population. The genetic data were then integrated with multiyear cone production records from the population of plus trees, thus facilitating the construction of a production population. Results A total of 80,539 SSR sites were identified among the 5,840,917 reads that were subjected to analysis. Notably, that the majority of these SSRs were dinucleotide to trinucleotide repeats, constituting 93.838% of the total. As the number of repeats increased, a gradual decline in the number of SSRs was observed. The most prevalent repetitive motif within the SSR loci was (AT/TA) n , representing 30% of all loci. A total of 1,933 SSR sites were selected for primer design, resulting in the successful formulation of 1,162 primer pairs, representing a success rate of 60.114%. Among these, 205 SSR primer pairs exhibited incomplete coverage of the SSR positions, whereas 79 primer pairs demonstrated specificity, as confirmed by e-PCR. Further analysis revealed 27 primer pairs suitable for amplification of the target fragments, nine of which exhibited polymorphisms. The mean number of alleles per primer ( N A ) was calculated to be 9.333, whereas the mean number of effective alleles ( N E ) was 4.032. The mean polymorphic information content (PIC) was 0.530. The Shannon index (I) for the plus tree population was 1.159, with observed heterozygosity ( H O ), expected heterozygosity ( H E ), and unbiased expected heterozygosity (µ H E ) values of 0.583, 0.530, and 0.533, respectively, and a fixation index (F) of −0.079. No statistically significant genetic differentiation was observed among the subgroups within the population, and the level of genetic diversity was comparable among subgroups delineated by different criteria. Significant differences in cone production were observed between the plus trees (P<0.01). A production population of 20 individuals was constructed via the simulated annealing algorithm, which exhibited a more reasonable mating system (F=−0.028) and demonstrated superior cone production compared with that of the plus tree population, with an increase of 79.6%. Conclusions The utilization of RAD-seq technology enables the development of SSR markers and the screening of polymorphic primers, thereby providing a robust tool for research pertaining to genetic diversity analysis and the identification of germplasm in Pinus koraiensis. The plus tree population in Xiaobeihu exhibits a considerable degree of genetic diversity, with no notable genetic differentiation. The construction of production populations via the developed SSR markers in combination with cone production from plus trees results in a reasonable population size and a mating system, thereby providing a scientific basis and technical support for the evaluation of Pinus koraiensis germplasm resources and advanced-generation improvement efforts. Furthermore, this study provides a reference and guidance for the development and application of SSR markers in other tree species.

Fraxinus mandshurica Rupr. is one of the main afforestation species in northeast China, and there is great demand for improved F. mandshurica varieties. The results of an investigation into and analysis of the growth traits of F. mandshurica provenances and families showed that there were significant differences in different periods. However, variations in growth traits decreased year by year. There was a significant negative correlation between tree heights of the provenances and sunshine hours in their areas of origin. The provenances of Xinglong, Hailin and Wuchang were selected based on the volume of 18-year-old trees. The average genetic gain from the selection of fast-growing provenances was 19.4%. Ten superior fast-growing families were selected. The average volume of the selected families was 22.6%, higher than that of all families. The correlation coefficient between heights at 6-year-old and at 18a was 0.838 for provenances, and between heights at 4-year-old and at 18-year-old was 0.303 for families. These results indicate that early selection for height in provenances or families could be performed at 6 years or 4 years, respectively. Early selection for DBH and volume in families could start at 8 years.

Suitable selection of endogenous controls is necessity for obtaining reliable qRT-PCR results. However, insufficient attention has been paid to the selection of endogenous controls for normalization in different stresses in plant, especially . In this study, we screened candidate internal reference genes for miRNA expression normalization after subjecting PEG-induced drought, NaHCO , or NaCl stress. Needles samples were collected at 0, 12, 24, 48 and 96 h after treatment. Four common housekeeping genes and 11 mature miRNAs that were found to be stably expressed in small RNA sequencing data of were selected as candidate internal reference genes. Their expression levels were assessed by qRT-PCR. GeNorm and NormFinder were used to analyse the stability of gene expression from the candidate internal reference genes. Finally, the expression of some miRNAs was analysed after stress. The results showed that miRNAs exhibited better expression stability compared to the analysed ncRNAs. Under PEG, NaHCO , or NaCl stress, miR83321, miR43169, and miR74940 were identified as the most suitable internal reference genes, respectively. Under PEG stress, the expression of most miRNAs reached its highest level at 12 h, showing a trend of first increasing expression and then decreasing. Similarly, under NaHCO and NaCl stress, the expression of most miRNAs reached its maximum levels at 48 h and 12 h, respectively. This indicates that most miRNAs more quickly respond to PEG and NaCl stress than NaHCO stress.

To improve the ecological and economic benefits of Korean pine (Pinus koraiensis), we analysed and evaluated its germplasm resources. This promotes in-depth research and utilisation of germplasm resources, providing excellent genetic resources for Korean pine breeding. We performed genetic analysis based on morphological and physiological traits and nuclear SSR molecular marker data was performed by collecting 314 clones (5 ramets of each clone) of Korean pine from eight (8) locations within the Korean pine range. The core collection underwent testing and evaluation for representativeness using variable rate (VR), coincidence rate (CR), variance difference percentage (VD), mean difference percentage (MD), Shannon index (I), and other indicators. The results indicated significant differences in morphological and physiological traits among the populations. All traits had a coefficient of variation (CV) greater than 10%, except for the water content of the needles (WC), which had an average CV of 17.636%. The populations showed high overall genetic diversity, with the HL (Helong) population exhibiting the highest genetic diversity, with an Ne (number of effective alleles), I, and He (expected heterozygosity) of 3.171, 1.103, and 0.528, respectively. Genetic variation mainly originated from individuals within populations, while the variation between populations was relatively small, at only 3%. The population did not exhibit any distinct subpopulation structures and was mainly derived from two admixed gene pools. Six core sets were obtained using different sampling strategies, and subset 6 was identified as the core collection, consisting of 114 individuals, representing a selection rate of 36.31%. In conclusion, the most appropriate method for constructing the core collection of Korean pines is the M-strategy (maximizing the number of alleles), based on both phenotypic and molecular data. The resulting core collection effectively represents the genetic diversity of the entire population effectively.

The miR169 family is one of the largest families of known miRNAs, which performs important functions in plant growth, development, and responses to biotic/abiotic stresses. However, its functions in response to abiotic stresses are still unclear in poplar. In present study, a total of 33 precursor MIR169s were identified from poplar and divided into 3 groups by evolutionary analysis and multiple sequence alignment, with the members in same group sharing similar motifs. Collinearity analysis revealed miR169s in other species that are homologous to poplar miRNAs. Cis-acting elements predication showed that miR169s may respond to ABA (Abscisic acid) and salt stress, which was verified by qRT-PCR. In addition, 12 pairs of miR169/target gene modules were identified by degradome sequencing and most of these modules responded to ABA and salt stress. Specifically, a part of miRNAs showed opposite expression trends with their targets at a certain period, demonstrating a repressive effect on the target genes. All the results suggest that miR169s perform important functions in response to abiotic stresses in poplar.

The lignified tissue in the secondary stem is the main source of wood. In this study, we applied RNA-Seq analysis to the poplar stems in three developmental stages, including primary stem (PS), transitional stem (TS), and secondary stem (SS), to identify a total of 2028 genes that were highly expressed in the SS. Gene annotation indicated that the functions of these genes are mainly involved in cell wall biosynthesis, xylem development, and programmed cell death (PCD) processes. Subsequently, we explored the expression pattern of these genes at various developmental stages in the horizontal direction of the wood by ASPwood. The expression of these genes was modularized and correlated with the percentage of lignified xylem, using weighted gene co-expression network analysis (WGCNA). Among the genes, as many as 690 were identified as directly associated with lignification in the SS. In addition, the gene promoter cis-elements and protein interactions were predicted by PlantRegMap and STRING, respectively. The results were introduced into a co-expression network to confirm their relationship. We eventually found 54 TFs dominating this network, of which ADOF1, ATMYB3, AtbZIP44 (Potri.005G231300), ANAC043, ATWRKY40, ATEBP (Potri.010G006800), ARF5, anac075, RAP2.1, ARF16, AT- HSFB3, Potri.014G050000 (from WRKY family), HAT22, AT-HSFB2B, and AtWRKY20 had extremely high connectivity, which may play an important role in the lignification of wood formation at secondary stages.