Improved analyses of genetic diversity and population structure by resolving polyploid SSR allele dosage uncertainty reveal the relationships between Chinese local apple varieties and M. sieversii

• The genetic analysis methods specifically tailored for polyploidy or mixed-ploidy populations to resolve allele dosage uncertainty in Malus polyploid SSR genotyping were first applied to the Chinese local apple varieties germplasm. • The Chinese cultivated species M. asiatica, M. asiatica var. rinki , and M. domestica subsp. chinensis as well as local varieties ‘Haihongguo’ all originated from M. sieversii . • The local varieties of southern Xinjiang ‘Kalaaerma’, ‘Kumoer’, and ‘Moluotuoyuzi’ were directly selected from M. sieversii due to their larger fruits and sweeter tastes. • Several local varieties of the Ili Valley region were derived from the introduced Russian cultivars ‘Ренет Ландсбергский’, ‘Ренет Бурхардта’, and ‘Титовка’. • M. domestica cultivars from Russia and Turkey were incompletely domesticated and maintained a closer genetic relationship with M. sieversii than those from Western Europe and North America. In this study, we applied genetic analysis methods specifically tailored for polyploidy and mixed-ploidy populations to evaluate 146 Malus accessions, primarily containing Chinese local apple varieties and M. sieversii . This new approach was implemented to enhance the accuracy and reliability of genetic diversity, structure, and differentiation analyses using SSR markers in Malus germplasm. We identified triploid M. asiatica individuals and both triploid and tetraploid M. sieversii individuals for the first time. Additionally, we found six synonymous groups in combination with an extensive evaluation of fruit characteristics. Bayesian-model-based STRUCTURE, non-model exploratory K-means clustering, and pairwise individual resemblance analyses consistently revealed five subgroups within this Malus germplasm collection. In conjunction with the genetic differentiation data of F ST and Jost’s D , we investigated their domestication history and genetic relationships with M. sieversii. M. domestica cultivars from Western Europe and North America evolved into a distinct species, whereas those from Russia and Turkey exhibited characteristics of incomplete domestication and maintained a closer relationship with M. sieversii . Chinese cultivated species, including M. asiatica, M. asiatica var. rinki , and M. domestica subsp. chinensis , as well as the local variety ‘Haihongguo’, all originated from M. sieversii . The local varieties ‘Kalaaerma’, ‘Kumoer’, and ‘Moluotuoyuzi’ from southern Xinjiang were directly selected and cultivated from M. sieversii forests. In contrast, local varieties from the Ili Valley region were derived from the introduced Russian cultivars ‘Ренет Ландсбергский’, ‘Ренет Бурхардта’, and ‘Титовка’. Many local varieties of Dongbei region were small hardy apples not indigenous but introduced from the Soviet Union. These findings obtained by the application of new methodologies provided valuable insights for the effective conservation and utilization of Chinese local apple varieties.