基于SSR标记对山梨猕猴桃与中华猕猴桃杂交群体的遗传多样性分析

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

摘要

【目的】山梨猕猴桃（ Actinidia rufa）具有极强的环境适应性，中华猕猴桃（ A. chinensis）拥有优异的果实品质，但两者亲缘关系较远。探究其种间杂交F1代群体的遗传结构、变异规律及亲本对F1代的遗传贡献，可为聚合抗逆性与优异果实品质的猕猴桃种质创新提供理论依据。【方法】利用40对高多态性SSR引物，对202份F1代单株及其亲本进行PCR扩增与毛细管电泳检测，利用GenoDive等软件计算遗传多样性参数，进行分子方差分析（AMOVA）、主成分分析（PCA）及杂交指数（hybrid index，h）计算，并筛选特异性引物构建DNA指纹图谱以鉴定杂种的真实性。【结果】40对SSR引物揭示了丰富的遗传变异，平均多态性信息含量（ PIC）高达0.631。F1群体表现出高水平的遗传多样性（ He=0.741， I=1.36）。群体观测杂合度（ Ho=0.813）显著高于预期杂合度（ He），表现出极显著的杂合性过剩（ P＜0.01），表明远缘杂交有效维持了基因组的杂合优势。AMOVA显示32.74%的变异来源于种间，遗传分化系数（Φst）为0.327，证明双亲间存在显著遗传分化。杂交指数（h≈0.35）、PCA及聚类分析一致揭示了F1群体遗传结构存在显著的父本（中华猕猴桃）偏倚现象。利用8对特异性SSR引物鉴定出F1代的真杂种率为93.07%~98.02%。【结论】利用SSR标记能够高效鉴定猕猴桃远缘杂交子代的真实性，研究明确了该组合F1群体强烈的父本遗传倾向。这一发现对制定回交育种策略及挖掘优异等位基因具有重要的指导意义。

Abstract

【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 resistance 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 exhibits high genetic diversity and significant heterozygote excess, showing a strong paternal genetic bias. This population is a valuable resource for aggregating stress resistance and superior fruit quality traits, providing important theoretical basis for kiwifruit molecular breeding and parent selection strategies.

关键词

山梨猕猴桃 / 中华猕猴桃 / 种间杂交 / SSR标记 / 遗传多样性

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基金资助

财政部和农业农村部：国家现代农业产业技术体系(CARS-26)

湖北省支持种业高质量发展资金项目(2023-2026)