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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.

Homeodomain-leucine zippers (HD-Zip) are plant-specific transcription factors that participate in different plant development processes and differentially regulate metabolic processes. LoHDZ2 is an HD-ZipII subfamily transcription factor gene that we identified from a transcriptomic analysis of Larix olgensis . To understand its function, we built a LoHDZ2 expression vector and then inserted it into tobacco by genetic transformation. Transgenic plants were identified at the DNA and RNA levels. Phenotypic index analysis of transgenic tobacco showed dwarfed growth with larger leaves and earlier flowering than the wild type. LoHDZ2 was expressed differently after hormone treatment with IAA, MeJA and 2,4-D. The results suggested that LoHDZ2 may respond to hormones and be involved in regulating growth and metabolism. These results helped us better understand the function of LoHDZ2 and provided a candidate gene for Larix olgensis molecular breeding.

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.

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.

Larch, a prominent afforestation, and timber species in northeastern China, faces growth limitations due to drought. To further investigate the mechanism of larch’s drought resistance, we conducted full-length sequencing on embryonic callus subjected to PEG-simulated drought stress. The sequencing results revealed that the differentially expressed genes (DEGs) primarily played roles in cellular activities and cell components, with molecular functions such as binding, catalytic activity, and transport activity. Furthermore, the DEGs showed significant enrichment in pathways related to protein processing, starch and sucrose metabolism, benzose-glucuronic acid interconversion, phenylpropyl biology, flavonoid biosynthesis, as well as nitrogen metabolism and alanine, aspartic acid, and glutamic acid metabolism. Consequently, the transcription factor T_transcript_77027, which is involved in multiple pathways, was selected as a candidate gene for subsequent drought stress resistance tests. Under PEG-simulated drought stress, the LoMYB8 gene was induced and showed significantly upregulated expression compared to the control. Physiological indices demonstrated an improved drought resistance in the transgenic plants. After 48 h of PEG stress, the transcriptome sequencing results of the transiently transformed LoMYB8 plants and control plants exhibited that genes were significantly enriched in biological process, cellular component and molecular function. Function analyses indicated for the enrichment of multiple KEGG pathways, including energy synthesis, metabolic pathways, antioxidant pathways, and other relevant processes. The pathways annotated by the differential metabolites mainly encompassed signal transduction, carbohydrate metabolism, amino acid metabolism, and flavonoid metabolism.

To date, there are few reports of the successful genetic transformation of larch and other conifers, mainly because it is difficult to transform and integrate exogenous genes. In this study, hybrid larch Larix kaempferi 3x Larix gmelinii 9 cones were collected on June 27, July 1, July 4, July 7 and July 16, 2017. Embryogenic callus induction was studied using a combination of different plant growth regulators and concentrations. The results showed that July 1 was the best stage; the highest induction rate was 10.83%, which cultured in BM medium (Button medium, which formula was listed in S1 Table) with 1.0 mg/L 2,4-D (2,4-dichlorophenoxyacetic acid) and 0.2 mg/L KT(kinetin). When cultured on a proliferation medium for 12 days, proliferation was the fastest, reaching 323.08%, which could also maintain the freshness and vitality. The suitable pre-culture medium for somatic embryogenesis was 1/4 BM medium containing 10 g/L inositol and 60 g/L sucrose. The combination of 45 mg/L ABA (abscisic acid) and 75 g/L PEG 4000 (Polyethyene glycol 4000) could promote the number of somatic embryos, and reached the maximum, 210 140 per 1 g FW. The genetic transformation was carried out by the Agrobacterium -mediated transformation method with embryogenic callus cultured for 12 days. The results showed the optimal OD 600 of the infection solution(suspension of A . tumefaciens ) was 0.5, co-culture time was 2 days, and screening concentration of Hyg (hygromycin B) was 4 mg/L. In this study, the transformation rate of resistance callus was 32.1%. It provides a reference for low genetic transformation efficiency of larch at present. This study could be beneficial for the innovation and breeding of larch by genetic engineering and provides a certain basis for rapid propagation of excellent larch germplasm resources and genetic engineering breeding of larch and other conifers.

Hybrid larch is the main timber and afforestation tree species in Northeast China. To solve the problem of rooting difficulties in larch cutting propagation, enzyme activity determination and transcriptome sequencing were carried out on the rooting tissues at five timepoints after cutting. peroxidase (POD), indole acetic acid oxidase (IAAO) and polyphenol oxidase (PPO) play important roles in the larch rooting process after cutting. A total of 101.20 Gb of clean data was obtained by transcriptome sequencing, and 43,246 unigenes were obtained after further screening and assembly. According to GO analysis and KEGG enrichment analysis, we think that plant hormones play an important role in the rooting process of larch stem cuttings. in the plant hormone signal transduction pathway, a larch gene c141104.graph_c0 that is homologous to the Arabidopsis AUX1 was found to be significantly up-regulated. We suggest that AUX1 may promote IAA transport in larch, thus affecting adventitious root development. According to the results of POD, PPO IAAO indexes and GO analysis, we think s1 and s2 periods may be important periods in the rooting process of larch stem cuttings, so we built a gene regulatory network, a total of 14genes, including LBD , NAC , AP2/ERF , bHLH and etc., may be important in different stages of cutting propagation. As the rooting rate after cutting inhibits the development of larch clone propagation, identifying the genes that regulate rooting could help us to preliminarily understand the molecular mechanism of adventitious root formation and select a better treatment method for cutting propagation.

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 Larch is an important timber tree species. The traditional methods of tree genetic breeding have been progressing slowly. It is necessary to carry out gene function analysis and genetically modified breeding research. The NAC transcription factor family is a plant-specific transcription factor family with various biological functions, as shown in recent research. However, there are few studies on the NAC gene among gymnosperm coniferous species. Results LoNAC3 with complete cds was identified and isolated from the cDNA of Larix olgensis based on transcriptome data. The cDNA length of LoNAC3 is 1185 bp, encoding 394 amino acids, with a conserved NAM domain located at the N-terminus, and subcellular localization in the nucleus. The results of real-time quantitative PCR analysis showed that at different growth stages and in different tissues of L. olgensis, the relative expression level of LoNAC3 was highest in the needles. After drought, salt, alkali stress and hormone treatment, expression was induced to different degrees. The expression level of LoNAC3 was significantly increased under drought and salt conditions. The relative expression level changed under methyl jasmonate (MeJA) and abscisic acid (ABA) treatment. By observing the phenotype of overexpressed LoNAC3 tobacco, it was found that overexpressed tobacco is shorter and blooms earlier than wild-type tobacco. Under abiotic stress, LoNAC3 overexpressed tobacco has lower germination rates and poorer growth status. Transgenic tobacco under stress treatment has a higher malondialdehyde (MDA) content than wild-type tobacco, while peroxidase (POD) activity is lower than wild-type tobacco. Conclusions Through the analysis of LoNAC3 sequence and promoter expression, it can be concluded that LoNAC3 is involved in the drought and salt stress response processes of L. olgensis , and is induced by ABA and MeJA expression. Overexpression of LoNAC3 leads to stunted tobacco growth and negatively regulates its tolerance to drought and salt stress through the reactive oxygen species pathway. The preliminary analysis of the expression pattern and function of the LoNAC3 can provide a theoretical basis and high-quality materials for genetic improvement of larch in later stages.

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.