Genome-shock deletion of a hybrid lethality gene breaks a reproductive barrier and facilitates speciation in Nicotiana

Shota Nagai^1,2Kenji Kawaguchi³Hiroki Itakura³Kaho Matsumoto⁴Takahiro Iizuka³Kosaku Kobayashi¹Kouki Nakata⁵Tetsuya Yamada⁵Wataru Marubashi⁶Masanori Yanase^1,3,4,7Toshinobu Morikawa^3,4Shuji Yokoi^1,3,4,7Takahiro Tezuka^1,3,4,7*

• ¹Graduate School of Agriculture, Osaka Metropolitan University, Sakai, Japan
• ²Osaka Furitsu Kankyo Norin Suisan Sogo Kenkyujo, Habikino, Japan
• ³Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Japan
• ⁴School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Japan
• ⁵Tokyo Noko Daigaku Nogakubu Daigakuin Nogaku Kenkyuin, Fuchu, Japan
• ⁶Meiji Daigaku Nogakubu Daigakuin Nogaku Kenkyuka, Kawasaki, Japan
• ⁷Education and Research Field, School of Agriculture, Osaka Metropolitan University, Sakai, Japan

Allopolyploidization plays an important role in the evolution of eukaryotes. Allopolyploids formed through interspecific hybridization and polyploidization undergo genetic and epigenetic changes in the early generations, known as 'genome shock'. However, reproductive isolation often prevents interspecific hybridization. The mechanism by which a reproductively isolated species breaks reproductive barriers and crosses with other species is largely unknown, despite its importance in speciation and evolution. Here, we report the ultrahigh-frequency appearance of viable hybrids that overcame hybrid lethality, a type of reproductive isolation, in crosses between Nicotiana tabacum and N. amplexicaulis. Lethal hybrids exhibited Type II hybrid lethality characterized by browning of hypocotyl and roots at 28°C, temperature sensitivity, and involvement of the Q chromosome from N. tabacum genome, indicating that N. amplexicaulis possesses the causal allele for hybrid lethality at the Hybrid Lethality A1 (HLA1) locus. Random amplified polymorphic DNA, amplified fragment length polymorphism, and methylation-sensitive amplified polymorphism analyses have detected genetic and epigenetic changes in viable and lethal hybrids, suggesting the occurrence of genome shock during interspecific hybridization. We found that many viable hybrids exhibited HLA1 locus deletion, indicating that it was the primary cause of overcoming hybrid lethality in these crosses. These findings demonstrate that genome shock-induced genetic changes promote the breakdown of reproductive barriers through the deletion of causal genes, providing insights into the mechanisms by which reproductively isolated species can overcome barriers and lead to the formation of new species.