Validation of kiwifruit sex molecular markers in Actinidia arguta

Abstract: 【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.