Pinus koraiensis (Sieb.et Zucc) is an economically and ecologically important tree species, naturally distributed in northeastern China. Conservation efforts and genetic improvement for this species began in the 1960s and 1980s, with the establishment of several primary seed orchards based on range-wide provenance evaluations. The original breeding objective was to improve growth and wood yield, but during the recent decade, it was redefined to include other traits, such as an enhancement of wood properties, seed oil content, cone yield, and the development of elite provenance with families, clones, and varieties with good tolerance to biotic and abiotic stresses. However, improvement processes are slow due to a long breeding cycle, and the number of improved varieties is still low. In this review, we summarize the recent progress in the selective improvement of P. koraiensis varieties, such as elite provenance, family, and clones, using various breeding procedures. We collate information on advances in the improvement of P. koraiensis, based on conventional breeding and molecular marker-assisted breeding methods; identify gaps in our understanding of the tree improvement processes; and propose future research directions, which will provide new insight for subsequent genetic breeding research on P. koraiensis.

Pinus koraiensis has significant economic and ecological value in Northeast China. However, due to the lack of suitable molecular markers, only a few available microsatellite markers were developed for further population genetics studies. In this study, for the first time we developed expressed sequence tag–simple sequence repeat (EST-SSR) markers from the cold-stressed transcriptome of P. koraiensis using Illumina Sequencing. We identified a total of 7,235 EST-SSRs from 97,376 sequences, and we tested their transferability among seven related Pinus species. The results showed that trinucleotides were the most abundant type of repeat (1287, 18.74%) excluding mononucleotides, followed by dinucleotides (1284, 18.7%) and tetranucleotides (72, 1.05%). The most dominant dinucleotides and trinucleotide repeat motifs were AT/AT (535, 7.79%) and AAT/ATT (103, 1.5%). The observed heterozygosity (Ho) and expected heterozygosity (He) ranged from 0.002 to 0.986 and 0.017 to 0.743, respectively, and the polymorphism information content (PIC) values and number of alleles (Na) varied from 0.029 to 0.794 and 2 to 23, respectively. A total of 8 natural P. koraiensis populations were divided into two main genetic clusters. Furthermore, nine of twenty polymorphic primer pairs were successfully amplified in seven Pinus species, and at least 80% of the successful P. koraiensis EST-SSR primers could be amplified in more than four species (16, 80%). Combined results for the development of EST-SSR markers in P. koraiensis and transferability among related species would contribute to improved studies on the genetic diversity and population structure in P. koraiensis and phylogenetic relationships among Pinus species. They would also provide a significant source for quantitative trait locus analysis.

The C-Repeat Binding Factor (CBF) gene family has been identified and characterized in multiple plant species, and it plays a crucial role in responding to low temperatures. Presently, only a few studies on tree species demonstrate the mechanisms and potential functions of CBFs associated with cold resistance, while our study is a novel report on the multi-aspect differences of CBFs among three tree species, compared to previous studies. In this study, genome-wide identification and analysis of the CBF gene family in Acer truncatum, Acer pseudosieboldianum, and Acer yangbiense were performed. The results revealed that 16 CBF genes (five ApseCBFs, four AcyanCBFs, and seven AtruCBFs) were unevenly distributed across the chromosomes, and most CBF genes were mapped on chromosome 2 (Chr2) and chromosome 11 (Chr11). The analysis of phylogenetic relationships, gene structure, and conserved motif showed that 16 CBF genes could be clustered into three subgroups; they all contained Motif 1 and Motif 5, and most of them only spanned one exon. The cis-acting elements analysis showed that some CBF genes might be involved in hormone and abiotic stress responsiveness. In addition, CBF genes exhibited tissue expression specificity. High expressions of ApseCBF1, ApseCBF3, AtruCBF1, AtruCBF4, AtruCBF6, AtruCBF7, and ApseCBF3, ApseCBF4, ApseCBF5 were detected on exposure to low temperature for 3 h and 24 h. Low expressions of AtruCBF2, AtruCBF6, AtruCBF7 were detected under cold stress for 24 h, and AtruCBF3 and AtruCBF5 were always down-regulated under cold conditions. Taken together, comprehensive analysis will enhance our understanding of the potential functions of the CBF genes on cold resistance, thereby providing a reference for the introduction of Acer species in our country.

Juglans mandshurica has strong freezing resistance, surviving temperatures as low as −40 °C, making it an important freeze tolerant germplasm resource of the genus Juglans. APETALA2/ethylene responsive factor (AP2/ERF) is a plant-specific superfamily of transcription factors that regulates plant development, growth, and the response to biotic and abiotic stress. In this study, phylogenetic analysis was used to identify 184 AP2/ERF genes in the J. mandshurica genome, which were classified into five subfamilies (JmAP2, JmRAV, JmSoloist, JmDREB, and JmERF). A significant amount of discordance was observed in the 184 AP2/ERF genes distribution of J. mandshurica throughout its 16 chromosomes. Duplication was found in 14 tandem and 122 segmental gene pairs, which indicated that duplications may be the main reason for JmAP2/ERF family expansion. Gene structural analysis revealed that 64 JmAP2/ERF genes contained introns. Gene evolution analysis among Juglandaceae revealed that J. mandshurica is separated by 14.23 and 15 Mya from Juglans regia and Carya cathayensis, respectively. Based on promoter analysis in J. mandshurica, many cis-acting elements were discovered that are related to light, hormones, tissues, and stress response processes. Proteins that may contribute to cold resistance were selected for further analysis and were used to construct a cold regulatory network based on GO annotation and JmAP2/ERF protein interaction network analysis. Expression profiling using qRT-PCR showed that 14 JmAP2/ERF genes were involved in cold resistance, and that seven and five genes were significantly upregulated under cold stress in female flower buds and phloem tissues, respectively. This study provides new light on the role of the JmAP2/ERF gene in cold stress response, paving the way for further functional validation of JmAP2/ERF TFs and their application in the genetic improvement of Juglans and other tree species.

The NAC (NAM, ATAF and CUC) gene family plays a crucial role in the transcriptional regulation of various biological processes and has been identified and characterized in multiple plant species. However, genome-wide identification of this gene family has not been implemented in Juglans mandshurica, and specific functions of these genes in the development of fruits remain unknown. In this study, we performed genome-wide identification and functional analysis of the NAC gene family during fruit development and identified a total of 114 JmNAC genes in the J. mandshurica genome. Chromosomal location analysis revealed that JmNAC genes were unevenly distributed in 16 chromosomes; the highest numbers were found in chromosomes 2 and 4. Furthermore, according to the homologues of JmNAC genes in Arabidopsis thaliana, a phylogenetic tree was constructed, and the results demonstrated 114 JmNAC genes, which were divided into eight subgroups. Four JmNAC gene pairs were identified as the result of tandem duplicates. Tissue-specific analysis of JmNAC genes during different developmental stages revealed that 39 and 25 JmNAC genes exhibited upregulation during the mature stage in walnut exocarp and embryos, indicating that they may serve key functions in fruit development. Furthermore, 12 upregulated JmNAC genes were common in fruit ripening stage in walnut exocarp and embryos, which demonstrated that these genes were positively correlated with fruit development in J. mandshurica. This study provides new insights into the regulatory functions of JmNAC genes during fruit development in J. mandshurica, thereby improving the understanding of characteristics and evolution of the JmNAC gene family.

Pinus koraiensis is a well-known precious tree species in East Asia with high economic, ornamental and ecological value. More than fifty percent of the P. koraiensis forests in the world are distributed in northeast China, a region with abundant germplasm resources. However, these natural P. koraiensis sources are in danger of genetic erosion caused by continuous climate changes, natural disturbances such as wildfire and frequent human activity. Little work has been conducted on the population genetic structure and genetic differentiation of P. koraiensis in China because of the lack of genetic information. In this study, 480 P. koraiensis individuals from 16 natural populations were sampled and genotyped. Fifteen polymorphic expressed sequence tag-simple sequence repeat (EST-SSR) markers were used to evaluate genetic diversity, population structure and differentiation in P. koraiensis. Analysis of molecular variance (AMOVA) of the EST-SSR marker data showed that 33% of the total genetic variation was among populations and 67% was within populations. A high level of genetic diversity was found across the P. koraiensis populations, and the highest levels of genetic diversity were found in HH, ZH, LS and TL populations. Moreover, pairwise Fst values revealed significant genetic differentiation among populations (mean Fst = 0.177). According to the results of the STRUCTURE and Neighbor-joining (NJ) tree analyses and principal component analysis (PCA), the studied geographical populations cluster into two genetic clusters: cluster 1 from Xiaoxinganling Mountains and cluster 2 from Changbaishan Mountains. These results are consistent with the geographical distributions of the populations. The results provide new genetic information for future genome-wide association studies (GWAS), marker-assisted selection (MAS) and genomic selection (GS) in natural P. koraiensis breeding programs and can aid the development of conservation and management strategies for this valuable conifer species.