性别分子标记在毛花猕猴桃中的通用性验证

General Validation of Kiwifruit Sex Molecular Markers in Actinidia eriantha Benth

【目的】猕猴桃是雌雄异株多年生木质藤本植物,其经济价值的主要载体为雌性植株的果实,将性别鉴定技术应用于早期实生苗对育种工作意义重大。目前已开发出可稳定鉴别中华猕猴桃和山梨猕猴桃种群性别的分子标记,可以检测出生物个体之间在核苷酸序列水平上的差异,极大降低了环境因素的干扰,验证这些标记在毛花猕猴桃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,从而在植株早期鉴定出其性别,以提高猕猴桃资源利用率。【方法】利用已知性别的10份江西野生毛花猕猴桃DNA样品作为材料,用聚丙烯酰胺凝胶电泳技术对PCR扩增产物进行检测,对退火温度进行多次筛选,确定最佳退火温度,对已报道的适用于某些猕猴桃品种的性别分子标记A001、A002、UDK96-009及UDK96-013等8个标记在毛花猕猴桃中的通用性进行验证。【结果】(1)8个分子标记中,A001、A002、UDK96-013和UDK96-019这4个标记均在毛花猕猴桃中能扩增出清晰明亮的多态性片段,但未能鉴别出有效的性别特异片段,因此不具备鉴定毛花猕猴桃性别的作用;(2)分子标记UDK96-009只在少量几个样品中扩增出较清晰的条带,但多态性较差,无法有效鉴定毛花猕猴桃的性别;(3)分子标记A003、UDK97-404和UDK97-408均无清晰的片段出现,同样无法鉴定出毛花猕猴桃的性别。【结论】本试验所使用的8个性别标记在毛花猕猴桃样品中扩增结果较差,未能在毛花猕猴桃中体现出良好的通用性,雌雄鉴定率较低,部分甚至无法扩增出清晰的条带,故这8个分子标记在鉴定毛花猕猴桃的性别上不具备通用性,无法用于毛花猕猴桃种质资源的性别鉴定。

[Objective]Kiwifruit is a kind of dioecious perennial woody vine,the vitamin C content in fruit of Actinidia eriantha Benth.is 3-4 times higher than that in A.chinensis Planch.The plants of Actinidia eriantha have high resistance to environmental stresses.Their fruits are rich in minerals with good flavor and taste.A.eri⁃antha is a kind of potential germplasm resource for high-quality new variety breeding.Dioecism is common for fruit trees,and the economic values of fruit trees are often greatly different due to the differences in sex.In production,kiwifruit fruits are produced by female plants,which are the main resources of economic value.However,male plants are mostly used as pairing trees for pollination and do not directly generate economic value.The juvenile period of kiwifruit is relatively long,generally 5-7a,while sexual identification is difficult in the juvenile period.It is significant to conduct early sex identification research by sex molecular markers for seedlings in the breeding process,which plays an important role in plantation configuration and breeding of new varieties.At present,molecular markers have been developed and that can be used to stably identify the sexes of specific populations of A.chinensis and Actinidia rufa,which also can be used to detect differences in nucleotide level among biological individuals,greatly reducing the interferences caused by environmental factors.Verifying the versatility of these molecular markers in the germplasm resources of A.eriantha can expand the application range of them in Actinidia species,thereby may be useful for identifying their sex in the early plant period and improving the utilization of kiwifruit resources.[Method]Eight kinds of reported molecular markers were used in this study.PCR products from DNA templates extracted from 10 different wild A.eriantha plants grown in Jiangxi with known sex were detected by polyacrylamide gel electrophoresis.In order to get clear and bright bands,the amplification temperature was screened for many times to find the possible most appropriate annealing temperature for these eight molecular markers in A.eriantha.[Result]The results showed that:(1)among the eight molecular markers,four markers,A001,A002,UDK96-013 and UDK96-019,could amplify clear and bright polymorphic fragments,the DNA ladder presented clear images,but effective sex-specific fragments were not identified,therefore,there did not function in identifying sex of kiwifruit;(2)the molecular marker,UDK96-009,only amplified clear bands in a few samples,but the polymorphism was poor,so it could not effectively identify the sex of A.eriantha;(3)the primers of molecular markers of A003,UDK97-404 and UDK97-408 did not amplify clear fragments,and it is also impossible to identify the sex of A.eriantha[Conclusion]The eight sex-related molecular markers used in this experiment had poor amplification results in A.eriantha samples,and failed to show good versatility,the accuracy of male and female identification was low,some of them even did not amplify clear bands,so these 8 molecular markers are not universal in identifying the sex of A.eriantha,and they can not be used for sex identification in A.eriantha germplasm resources.Because of the complicated genetic background of kiwifruit,the reasons for the poor commonality of sex markers are unclear,and further research is needed to explain these results.It is speculated that the reason for this is that the sex determination of kiwifruit complies with Mendelian inheritance laws and is characterized by single gene control.However,the existing research is insufficient.Further study is needed to find means for sex determination of kiwifruit.