Analysis of genetic diversity of Actinidia rufa and Actinidia chinensis hybrids using SSR markers

【Objective】Kiwifruit (Actinidia spp.) is a globally important economic fruit crop with a diverse genetic background. Actinidia rufa is characterized by strong resistance to biotic and abiotic stresses, particularly environmental adaptability, whereas Actinidia chinensis is widely cultivated for its large fruit size, excellent flavor, and high commercial value. Interspecific hybridization between these two distantly related species represents a critical pathway for breeding novel cultivars that combine stress re-sistance with superior fruit quality (“wide crossing”). However, due to the complexity of the Actinidia genome and potential reproductive barriers in distant hybridization, the genetic architecture, inheritance patterns, and parental contribution in their F1 progeny remain largely unclear. Understanding whether the offspring genetically incline towards the maternal or paternal parent is crucial for designing subsequent breeding strategies, such as backcrossing schemes. This study aims to evaluate the genetic diversity, population structure, and authenticity of 202 F1 individuals derived from a cross between A. rufa (female) and A. chinensis (male) using Simple Sequence Repeat (SSR) markers. The specific goals are to validate the efficiency of SSRs for hybrid identification, quantify the level of genetic differentiation between parents, and elucidate the phenomenon of genetic segregation distortion or parental bias in the hybrid population.【Methods】A total of 202 F1 individuals and their parents were used as experimental materials. Genomic DNA was extracted from young leaves. A rigorous screening process was conducted to select 40 pairs of highly polymorphic SSR primers from a larger pool of candidate markers. These primers were used for PCR amplification, and the products were detected using high- resolution capillary electrophoresis to ensure accurate allele sizing. Genetic diversity parameters, including the Number of Alleles (Na), Effective Number of Alleles (Ne), Observed Heterozygosity (Ho), Expected Heterozygosity (He), Shannons Information Index (I), and Polymorphism Information Content (PIC), were calculated to assess the variability within the population. Analysis of Molecular Variance (AMOVA) was performed to partition the genetic variance among and within populations, and the genetic differentiation coefficient (Φst) was calculated to quantify the divergence between the parental species. To visualize the genetic structure and relationships, Principal Component Analysis (PCA) and UPGMA (Unweighted Pair Group Method with Arithmetic Mean) cluster analysis were conducted. Furthermore, the Hybrid Index (h) was calculated for each individual to quantify the genomic contribution of each parent, ranging from 0 to 1. Finally, a subset of 8 pairs of specific, complementary SSR primers was selected to identify true hybrids and construct DNA fingerprints for the population.【Results】The molecular analysis revealed that the 40 selected SSR primers were highly informative, detecting a wealth of genetic variation within the population with an average Polymorphism Information Content (PIC) of 0.631. The F1 population exhibited a high level of genetic diversity, characterized by a mean Expected Heterozygosity (He) of 0.741 and a Shannon’s Information Index (I) of 1.36. A significant finding was the phenomenon of "Heterozygote Excess", where the Observed Heterozygosity (Ho = 0.813) was significantly higher than the Expected Heterozygosity (He = 0.741, P＜0.01), suggesting that the interspecific hybridization effectively broke the linkage drag or inbreeding depression often found in intraspecific crosses. AMOVA results indicated that 32.74% of the total genetic variation occurred among populations, with a genetic differentiation coefficient (Φst) of 0.327, confirming that A. rufa and A. chinensis are genetically distinct species with significant allelic divergence. Crucially, the analysis of genetic structure revealed a striking pattern of paternal bias. The Hybrid Index (h ≈ 0.35), together with PCA and cluster analyses, consistently revealed a significant paternal bias (toward A. chinensis) in the genetic structure of the F1 generation—a pattern markedly different from the maternal bias commonly observed in species such as tea plants. This phenomenon was distinct from the maternal inheritance patterns that were often reported in other woody plants. Additionally, using the 8 specific SSR primer pairs, the study successfully distinguished true hybrids from potential selfs or outcrosses, with a true hybrid rate between 93.07% and 98.02% . DNA fingerprints containing parent- specific loci were constructed for the authenticated hybrids.【Conclusion】This study successfully demonstrates the utility of SSR markers for the rapid and accurate identification of interspecific hybrids in Actinidia. The F1 population derived from A. rufa × A. chinensis is characterized by high genetic diversity and significant heterozygote excess, providing abundant variation for breeding selection. Most notably, the study reveals a strong paternal genetic tendency (bias towards A. chinensis) in the F1 generation. This finding implies that while the hybrids successfully inherit the desired quality traits from the paternal A. chinensis, there is a risk of losing the resistance traits from the maternal A. rufa due to segregation distortion. Therefore, this study provides a critical molecular theoretical basis for future breeding strategies: to combine high quality with high resistance, larger population sizes may be needed to find the rare individuals with balanced inheritance, or specific backcross strategies to the maternal parent may be required to recover the resistance genes. The constructed DNA fingerprints will serve as a permanent record for germplasm protection and management.