利用多分子标记分析‘和平红阳’猕猴桃的性别差异

Analysis of gender difference of Actinidia chinensis 'Hepinghongyang' using multiple-markers

【目的】分子生物学技术是鉴定植物种质多样性的常规方法,由于不同的核酸类型和不同的DNA片段其保守性不一致,有效选择一种合适的分子标记技术或结合多个分子标记物可以更加客观和高效地分析种质多样性。【方法】通过总核酸提取法提取‘和平红阳’猕猴桃核基因组DNA,叶绿体和线粒体DNA以及总RNA,并以SSR、RAPD、ddRT-PCR、小片段RNA分子标记以及叶绿体和线粒体DNA条形码等5种分子标记方法对‘和平红阳’猕猴桃雄株与雌株进行差异性分析。【结果】利用总核酸提取法可以很好获得‘和平红阳’猕猴桃核基因组DNA,核外叶绿体和线粒体DNA以及50bases以上的总RNA;‘和平红阳’猕猴桃雄、雌株的SSR、RAPD、ddRT-PCR和小片段RNA分子标记的差异性分别为82.7%、63.8%、50.4%和0,说明不同的分子标记物差异不同;‘和平红阳’猕猴桃雄雌株间的3个叶绿体和线粒体DNA条形码psbA、rbcL和nad1的相似度和系统树距离分别为99.19%、0.00706,100%、0和99.37%、0.00377,说明不同的DNA条形码在雄雌株间具有差异性。【结论】‘和平红阳’猕猴桃不同核酸片段变异速率不一致,在实际应用过程中可以根据群体亲缘关系大小来选择一种合适的分子标记物。

[Objective]Molecular biological technique is a conventional method which is used to identify the germplasm diversity. Due to the differences of conservation inconsistency among the deferent nucleic acid types and DNA fragments, it is more objective and efficient to analyze the germplasm diversity by choosing an appropriate molecular marker technology or combining multiple molecular markers. [Method] The nuclear genome DNA, chloroplast and mitochondrial DNA, and total RNA of A. chinensis ＇Hep- inghongyang＇ were extracted by total nucleic acid extraction method, and the genetic difference between the male and female of A. chinensis ＇Hepinghongyang＇ was analyzed by SSR, RAPD,ddRT-PCR, short RNAs and DNA barcoding. [Result]The results showed that the nuclear genomic DNA, chloroplast and mitochondrial DNA, and total RNA above 50 bp could be extracted from A. chinensis ＇ Hepinghongyang＇ by total nucleic acid method; the difference of SSR,RAPD,ddRT-PCR,short RNAs between male and female of A. chinensis ＇ Hepinghongyang＇ were 82.7%, 63.8%, 50.4% and 0 respectively, indicating vari- ous molecular markers are different in PPB ; the similarity and phylogeny tree distance of psbA and rbcL in chloroplast, and nadl in mitochondrial were 99.19% ,0.00706, 100% ,0 and 99.37% ,0.00377 respec- tively, indicating that there were diversiform in different barcordings between male and female of A. chi- nensis ＇Hepinghongyang＇. [Conclusion]The studies suggested that the mutation rates of different nucleicacid fragments of A. chinensis ＇Hepinghongyang＇ were inconsistent, and the suitable molecular markers should be selected according to the kinship of groups during practical application.The study provided a ba- sis for multiple-markesr analysis of kiwifruit breeding program and germplasm resources by using five molecular markers to analyze the gender genetic difference between male and female of A. chinensis ＇ Hepinghongyang＇.