猕猴桃性别分子标记在软枣猕猴桃中的通用性验证

Validation of kiwifruit sex molecular markers in Actinidia arguta

【目的】猕猴桃是功能性的雌雄异株植物,在育种过程中进行实生苗早期性别鉴定研究具有重要意义。目前已有关于猕猴桃属植物早期性别鉴定分子标记开发的报道,用于特定种群实生苗童期的性别鉴定。验证这些标记在其他来源种质资源中的通用性可扩大这些标记在猕猴桃属植物中的应用范围。【方法】利用已知性别的64份软枣猕猴桃材料,采用普通PCR扩增的方法以及聚丙烯酰胺凝胶电泳技术,对已报道的两组猕猴桃性别分子标记A001、A002和A003,SmX和SmY1的通用性进行检验。【结果】(1) A001和A002在软枣猕猴桃中均能扩增出多态性片段,且条带清晰明亮,但未出现性别特异片段;(2) A003的PCR扩增结果凝胶图像中,没有清晰的片段出现;(3)在对已知性别的软枣猕猴桃资源的验证试验中,SmX的扩增结果图片显示,所扩增的条带未表现出雌雄特异性,准确率很低,且条带微弱;(4) SmY1在软枣猕猴桃中能够扩增出多态性片段,但未出现性别特异的片段。【结论】在本研究所用的软枣猕猴桃种质资源中,两组标记均未表现出良好的通用性,其性别扩增结果的雌雄鉴别率很低,甚至不能扩增出清晰的片段,故不能用于本研究所用软枣猕猴桃种质资源的性别鉴定。

【Objective】The kiwifruit varieties currently cultivated and grown mainly come from Actinidia chinensis Planch.In the production,Actinidia arguta(kiwiberry)is a new type of kiwifruit and it develops rapidly and becomes more and more popular among consumers in recent years.Actinidia arguta has begun to receive extensive attention in the kiwifruit community.Kiwifruit is a functional dioecious plant,and its sex differentiation is controlled by sex-determining genes,which are located in a pair of chromosomes like the XY/XX system with the Y chromosome specific in male individuals.Gender differentiation is independent of plant ploidy level.Generally,the juvenality of kiwifruit plant is about 5-7 years.Due to the complicated genetic background,male individuals of the seed offspring seem to be more than the female ones.However,the male and female individuals of kiwifruit seedlings are difficult to distinguish by the morphological features in the juvenile phase.At present,there have been some reports about the development of molecular markers for sex identification in kiwifruit,but these markers are effective only in A.chinensis Planch.or the restricted hybrid progeny.The versatility of the reported markers in the sex identification of A.arguta is still unclear.Some researches on the sex identification of A.arguta have been reported although its accuracy is still not satisfactory for the commercial use.【Methods】We chose two sets of reported molecular markers:SSR markers A001,A002 and A003,and SCAR markers SmX and SmY1,and we randomly selected 64 accessions of A.arguta,whose genders were already known as research materials.Young leaves were taken from the healthy annual branches of mature A.arguta plants of similar age for genome DNA extraction.The accuracy of each marker was validated by ordinary PCR technique.we set 8 annealing temperature gradients to screen the appreciate system of each pair of primer before all the A.arguta materials were tested.To make sure that the results of PCR experiments are reliable,the validation experiments of each molecular marker were repeated at least three times.【Results】The results showed that:(1)The three SSR markers,A001,A002 and A003,performed differently in A.arguta samples.There were polymorphic fragments amplified by A001 and A002 in the products of the A.arguta samples,but result pictures did not showed gender specificity.Marker A003 did not amplify polymorphic fragments in A.arguta.(2)When SmY1 was tested in the samples of A.arguta,there were some polymorphic fragments amplified,but the fragments showed in the pictures were irregular and they had no any difference between the males and the females;(3)when SmX was identified in A.arguta samples,there was no polymorphic fragment while the DNA ladder presented a clear image.【Conclusion】The above results indicated that,in A.arguta,the two sets of molecular markers could not be used for sex identification of this species.Because of the complicated genetic background of kiwifruit,the reason of the result is still vague and worthy of further investigation.The author speculated that the position or sequence of the sex-specific region of A.arguta might be different from that of A.chinensis Planch.