The formation of diploid gametes through chromosome doubling is a major mechanism of polyploidization, diversification, and speciation in plants. Unfavorable climate conditions can induce or stimulate the production of diploid gametes during meiosis. Here, we demonstrated that heat shock stress (38°C for 3 or 6 h) induced 2n pollen formation, and we generated 42 triploids derived from heat shock-induced 2n pollen of Populus canescens. Meiotic analysis of treated pollen mother cells revealed that induced 2n pollen originated from the complete loss of meiosis II (MII). Among the 42 triploids, 38 triploids derived from second division restitution (SDR)-type 2n pollen and 4 triploids derived from first division restitution-type 2n pollen were verified using simple sequence repeats (SSR) molecular markers. Twenty-two differentially expressed genes related to the cell cycle were identified and characterized by expression profile analysis. Among them was POPTR_0002s08020g (PtCYCA1;2), which encodes a type A Cyclin CYCA1;2 that is required for the meiosis I (MI) to MII transition. After male flower buds were exposed to heat shock, a significant reduction was detected in PtCYCA1;2 expression. We inferred that the failure of MI-to-MII transitions might be associated with downregulated expression of PtCYCA1;2, leading to the formation of SDR-type 2n pollen. Our findings provide insights into mechanisms of heat shock-induced 2n pollen formation in a woody plant and verify that sensitivity to environmental stress has evolutionary importance in terms of polyploidization.

Genetic diversity analysis provides the scientific basis for the preservation, evaluation, and utilization of the germplasm resources of tree species. We explored the genetic diversity and structure of Populus tomentosa elite trees in North China using 13 nuclear microsatellite markers. We compared nine groups of accessions including 20 originating from Beijing (BJ), 122 from Hebei (HB), 20 from Shandong (SD), 113 from Henan (HN), 270 from Shanxi (SX), 54 from Shaanxi (SAX), 8 from Gansu (GS), 10 from Anhui (AH), and 6 from Jiangsu (JS). All of the studied primer pairs were polymorphic and generated 125 alleles. Analyses of molecular variance revealed that 79%, 14%, and 8% of the total variation was due to variations within the individual, among individuals, and among populations, respectively. Based on principal coordinate and STRUCTURE cluster analyses, individuals distributed in the southern region (HN, SAX, AH, and JS) were roughly classified into one group, while those distributed in the northeastern region (BJ, HB, and SD) and northwestern regions (SX) were separately divided into one group each. Moreover, the northwestern region included two-thirds of the SX trees, and the remainder were in the northeast region. By analyzing genetic diversity and structure within populations, individuals with different genetic backgrounds were screened for constituent training populations (TRS), including broad allelic variation for related traits. This ensures that the genomic prediction model can accurately capture genetic effects and provide reliable predictions across a broad spectrum of genetic backgrounds. Therefore, our results will benefit genome breeding technology.