Cold-adaptive evolution at the reproductive stage in Geng/japonica subspecies reveals the role of OsMAPK3 and OsLEA9.

Cold stress at the reproductive stage severely affects the production and geographic distribution of rice. The Geng/japonica subpopulation gradually developed stronger cold adaptation than the Xian/indica subpopulation during the long-term domestication of cultivated rice. However, the evolutionary path and natural alleles underlying the cold adaptability of intra-Geng subspecies remain largely unknown. Here, we identified MITOGEN-ACTIVATED PROTEIN KINASE 3 (OsMAPK3) and LATE EMBRYOGENESIS ABUNDANT PROTEIN 9 (OsLEA9) as two important regulators for the cold adaptation of Geng subspecies from a combination of transcriptome analysis and genome-wide association study. Transgenic validation showed that OsMAPK3 and OsLEA9 confer cold tolerance at the reproductive stage. Selection and evolution analysis suggested that the Geng version of OsMAPK3 (OsMAPK3 ) directly evolved from Chinese Oryza rufipogon III and was largely retained in high-latitude and high-altitude regions with low temperatures during domestication. Later, the functional nucleotide polymorphism (FNP-776) in the Kunmingxiaobaigu and Lijiangxiaoheigu version of the OsLEA9 (OsLEA9 ) promoter originated from novel variation of intra-Geng was selected and predominantly retained in temperate Geng to improve the adaptation of Geng together with OsMAPK3 to colder climatic conditions in high-latitude areas. Breeding potential analysis suggested that pyramiding of OsMAPK3 and OsLEA9 enhanced the cold tolerance of Geng and promotes the expansion of cultivated rice to colder regions. This study not only highlights the evolutionary path taken by the cold-adaptive differentiation of intra-Geng, but also provides new genetic resources for rice molecular breeding in low-temperature areas.