Cytological mechanisms underlying fertility differences in allotetraploid tree peonies

Tetraploidization is a critical pathway for fertility restoration in inter-subsectional hybrid tree peonies ( Paeonia × lemoinei ). However, mechanisms underlying the male sterility or reduced fertility observed in some allotetraploid tree peonies remain unclear. In this study, three allotetraploid tree peonies (07-LB-30, 07-LB-22, and 07-LB-56) with different fertility levels were investigated by observing meiosis behavior and anther development, characterizing pollen morphology and viability, and analyzing chromosomal composition via genomic in situ hybridization (GISH). We found that the fertility differences among the three materials did not align with their overall frequency of meiotic irregularities, suggesting that meiotic disturbance could not necessarily lead to fertility defects in these allotetraploids. Despite forming 17.57% normal tetrads, 07-LB-22 exhibited defective tapetum development and subsequent thorough pollen abortion, which more possibly result in its male sterility. Similarly, although 07-LB-56 generated a moderate proportion (38.90%) of normal tetrads, its severe chromosomal pairing disorders, which may lead to a high frequency of aneuploid gametes, are supposed to account for its reduced fertility. GISH revealed that 07-LB-56 possesses an AAAB type chromosomal composition, distinct from the near-AABB configuration of the other two materials. This asymmetric subgenomic composition could cause genomic imbalance, thereby giving rise to pairing failure and reduced fertility. The AAAB-type allotetraploid tree peony might originate from backcrossing between inter-subsectional hybrid tree peonies ( P. × lemoinei , AABB or AB) and common tree peonies ( P. suffruticosa , AA), suggesting a potential pathway for tetraploidization in tree peonies. This study elucidated the mechanisms underlying fertility differences in three allotetraploid tree peonies, and proposed a backcrossing pathway for tetraploidization, providing valuable theoretical bases for polyploid breeding in tree peonies.