新疆野苹果S等位基因的遗传多样性和进化研究

Malus sieversii (Ledeb.) Roem. is not only the wild ancestor of Malus × domestica but also an important wild germplasm resources. There were plenty of studies on S-alleles and S-genotypes of Malus × domestica, while the current project focuses on the S alleles of Malus sieversii. The nucleotide sequences of S-alleles of Malus sieversii would be obtained through PCR amplification, followed by cloning and DNA sequencing, and then the genetic diversity of S-alleles could be estimated for wild populations of Malus sieversii. Through comparison with S-alleles found from Malus × domestica, the S-alleles which were specific to Malus sieversii could be picked out, and sequence specific primers would be developed for rapid identification of S-genotypes of Malus sieversii. Based on the richness of S-alleles and distribution of S-genotypes, the mating pattern of Malus sieversii under natural condition could be inferred, and the probable mating limitation, due to self-incompatibility and the lack of compatible pollens resulted from population decline could be explored. Lastly, the evolutionary properties of S-allele would be investigated based on population genetic analyses. Our project extend the S-allele researches from Malus × domestica to its wild ancestor, Malus sieversii, and it would be the first molecular population genetic studies of S-allele for species from genus Malus. Furthermore, the sequences of S-alleles and constitution of S-genotypes collected from Malus sieversii should be valuable for germplasm evaluation, utilization and protection of this important wild fruit tree resource.

新疆野苹果不仅是栽培苹果的野生祖先，也是重要的野生果树资源。本项目拟以栽培苹果S等位基因和S基因型的研究为基础，利用保守引物扩增和克隆测序的方法获得新疆野苹果的S基因序列，然后估计S等位基因在新疆野苹果居群中的遗传多样性；并且根据栽培苹果的S基因序列确定新疆野苹果特有的S等位基因，进而设计序列特异引物实现新疆野苹果S基因型的快速鉴定。同时计算亲子两代S等位基因丰富度和S基因型分布，由此确定新疆野苹果的交配模式，并探讨新疆野苹果是否由于自交不亲和的繁育属性，以及居群规模缩小导致亲和花粉不足而遭遇配偶限制的问题；最后研究S等位基因在自然野生居群中的进化规律。本项目将S等位基因与自交不亲和性的研究拓展到栽培苹果的野生祖先－新疆野苹果上，获取的S等位基因和S基因型数据不仅填补了苹果属植物S基因分子居群遗传学和进化研究的空白，也为新疆野苹果种质资源的科学利用和保护提供了重要信息。

本项目圆满完成了预期目标。我们对新疆野苹果S等位基因的遗传多样性进行了详细检测。通过使用3对保守引物进行聚合酶链式反应以及分子克隆与测序，我们检测了99个新疆野苹果个体的S等位基因。同时为了比较S基因与中性基因进化特性差异，我们还测定了8个独立进化的中性基因。采用系统发育、分子进化和群体遗传学方法分析S基因和中性基因的核苷酸序列数据，得到下列重要结果。新疆野苹果仅有14种S等位基因，显著少于栽培苹果（32）和欧洲野苹果（38）中的S等位基因。分子进化分析检测到S等位基因中大约15%的氨基酸位点受到正选择，这与其参与花粉-柱头亲和性识别的功能吻合。群体遗传学分析揭示S等位基因的遗传多样性显著高于中性基因，但群体遗传分化水平却明显低于中性基因，证明S位点在新疆野苹果中受到平衡选择作用。配偶可得性分析显示新疆野苹果群体不存在配偶限制。本项目全面系统的研究了栽培苹果及其野生亲本新疆野苹果与欧洲野苹果S等位基因的遗传多样性和谱系关系，开发了能有效鉴定S位点基因型的保守引物，上述研究成果不仅有助于更有效的保护和利用新疆野苹果这一珍贵野生种质资源，同时也证明S等位基因能用于研究植物种群动态的进化过程。