Identification of S-RNase genotype and analysis of its origin and evolutionary patterns in Malus plants

Identification of the S genotype of Malus plants will greatly promote the discovery of new genes, the cultivation and production of apple, the breeding of new varieties, and the origin and evolution of self-incompatibility in Malus plants. In this experiment, 88 Malus germplasm resources, such as Aihuahong, Xishuhaitang, and Reguanzi, were used as materials. Seven gene-specific primer combinations were used in the genotype identification. PCR amplification using leaf DNA produced a single S-RNase gene fragment in all materials. The results revealed that 70 of the identified materials obtained a complete S-RNase genotype, while only one S-RNase gene was found in 18 of them. Through homology comparison and analysis, 13 S-RNase genotypes were obtained : S1S2 (Aihuahong, etc.), S1S28 (Xixianhaitang, etc.), S1S51 (Hebeipingdinghaitang), S1S3 (Xiangyangcundaguo, etc.), S2S3 (Zhaiyehaitang, etc.), S3S51 (Xishanyihao), S3S28 (Huangselihaerde, etc.), S2S28 (Honghaitang, etc.), S4S28 (Bo 11), S7S28 (Jiuquanshaguo), S10Se (Dongchengguanshisanhao), S10S21 (Dongxiangjiao) and SeS51 (Xiongyuehaitang). Simultaneously, the frequency of the S gene in the tested materials was analyzed. The findings revealed that different S genes had varying frequencies in Malus resources, as well as varying frequencies between intraspecific and interspecific. S3 had the highest frequency of 68.18%, followed by S1 (42.04%). In addition, the phylogenetic tree and origin evolution analysis revealed that the S gene differentiation was completed prior to the formation of various apple species, that cultivated species also evolved new S genes, and that the S50 gene is the oldest S allele in Malus plants. The S1, S29, and S33 genes in apple-cultivated species, on the other hand, may have originated in Malus sieversii, Malus hupehensis, and Malus kansuensis, respectively. In addition to Malus sieversii, Malus kansuensis and Malus sikkimensis may have also played a role in the origin and evolution of some Chinese apples.