Validation of Some Molecular Markers in Sex Determination in Different Kiwifruit Genotypes from Open Pollination

Kiwifruit is one of the important exportable horticultural crops in Iran. international customers tend to buy novel kiwifruit with different flesh and taste has increased. Therefore, for persistence in exporting and success in export rank of Iranian kiwifruit, it is necessary to have new cultivars. One of the main limitations in kiwifruit breeding program, juvenility period is long time and it is dioecy which there is no distinguishable differences between male and female plants until flowering time. Therefore, sex determination in seedling and separation male and female plants as soon as possible is very important for accelerating kiwifruit breeding. In this study, validation of some molecular sex related markers for determination males and female plants results from open pollination.

In this study, we used two different yellow flesh kiwifruit genotypes (known as Khorshid and Navab) and one red flesh genotype, which all were belong to Actinidia chinensis with diploid. Extraction of genomic DNA from young leaves was carried out by CTAB method with few modifications. spectrophotometer (Biophotometra, Plus Eppendorff) is used for determine the exact concentration of each sample DNA, and DNA concentration was measured at a wavelength of 260 nm. Polymerase chain reaction (PCR) was performed in a volume of 10 μL for each sample. For each primer program (PCR), agarose gel concentration and optimum loading time was optimized. Finally, the validation of some sex related markers includes 3 SCAR markers (SmY, SmX, SmY1) and 3 SSR markers (A00I, A00II, A00II) in the DNA extracted from the leaves of the tested genotypes was investigated.

The results of current study showed that among different SCAR and SSR markers which previously reported, some of them were not able to separate correctly male and female seedlings. Among three SCAR markers (SmY, SmX and SmY1) were evaluated, just two markers; SmY1 (770 bp) and SmX (950bp) markers could able to separate correctly male (without band pattern in female) and female (without band pattern in male) kiwifruit seedlings respectively. According to polymorphic bands were obtained from three tested SSR markers (A00I, A00II, A00II) for determination males and females plants, only A00II with specific bands 232bp in male and 219bp in female plants could able to separate kiwifruit seedlings. The two A00I and A00III markers exhibited the similar banding patterns in males and females which were not able to separate individuals from gender.

Overall, the results of this study showed that simultaneously application of SmY1, SmX and A00II are able to separate correctly male and female seedlings of kiwifruit during the early juvenile phase.