Grafting, cutting, and pruning are important horticultural techniques widely used in the establishment of clonal forestry. After the application of these techniques, some properties of the plants change, however, the underlying molecular mechanisms are still unclear. In our previous study, 27 age-related transcripts were found to be expressed differentially between the juvenile vegetative (1- and 2-year-old) and adult reproductive (25- and 50-year-old) phases of Larix kaempferi. Here, we re-analyzed the 27 age-related transcripts, cloned their full-length cDNA sequences, and measured their responses to grafting, cutting, and pruning. After sequence analysis and cloning, 20 transcription factors were obtained and annotated, most of which were associated with reproductive development, and six (LaAGL2-1, LaAGL2-2, LaAGL2-3, LaSOC1-1, LaAGL11, and LaAP2-2) showed regular expression patterns with L. kaempferi aging. Based on the expression patterns of these transcription factors in L. kaempferi trees subjected to grafting, cutting, and pruning, we concluded that (1) cutting and pruning rejuvenate the plants and change their expression, and the effects of cutting on gene expression are detectable within 14 years, although the cutting seedlings are still maturing during these years; (2) within three months after grafting, the rootstock is more sensitive to grafting than the scion and readily becomes mature with the effect of the scion, while the scion is not readily rejuvenated by the effect of the rootstock; and (3) LaAGL2-2 and LaAGL2-3 are more sensitive to grafting, while LaAP2-2 is impervious to it. These findings not only provide potential molecular markers to assess the state of plants but also aid in studies of the molecular mechanisms of rejuvenation.

Key message: Expression analysis of Larix kaempferi mature miR171s and their primary transcripts and target gene LaSCL6 during somatic embryogenesis revealed the transcriptional and post-transcriptional regulation of the miR171-LaSCL6 module. Somatic embryogenesis provides a useful experimental system for studying the regulatory mechanisms of plant development. The level and activity of microRNA171 (miR171) fluctuate during somatic embryogenesis in Larix kaempferi, but the underlying mechanisms are still unclear. Here, in L. kaempferi we identified five members of the miR171 family, which cleave LaSCL6 mRNA at different sites. In addition, we improved the method of measuring miRNA activity in a more direct way. Furthermore, we measured the expression patterns of mature miR171s and their primary transcripts during somatic embryogenesis in L. kaempferi and found that their patterns differed, indicating that the transcription of MIR171 genes and the subsequent cleavage of their intermediate products are regulated. Taken together, our findings not only offer a means to study the regulation of miRNA activity, but also provide further insight into the regulation of L. kaempferi somatic embryogenesis by miR171-LaSCL6.

Simple sequence repeats (SSRs) are widely used as markers for the assessment of genetic diversity and marker-assisted breeding. In a previous study, two SSRs (GCA and CCA), were found in the genomic sequence of Larix (La) SCL6, which plays important roles in the growth and development of Larix kaempferi (Lamb.) Carr. In this study, we analyzed the polymorphisms of these two SSRs in the L. kaempferi population. We found that each SSR had five different polymorphisms, among which (GCA)₇ and (CCA)₇ were predominant. In addition, 12 haplotypes were detected, with (GCA)₇(CCA)₇ having the highest frequency. Furthermore, we detected the haplotypes of LaSCL6 in mature trees and their seeds and analyzed the relationships between parents and offspring. The expression patterns of five LaSCL6 alleles were analyzed and they showed balanced expression during vegetative development. Taken together, these findings not only provide more genetic information on LaSCL6, but also provide a candidate marker for genetic studies and breeding.