การจำแนกเพศของอินทผลัม (Phoenix dactylifera L.) ด้วยการวิเคราะห์ไฮเรโซลูชันเมลติง

Abstract

Date palm (Phoenix dactylifera L.) is one of the economic crops that tolerate to high temperatures. Dates contain a relatively high sugar content and are rich in a variety of nutrients. Recently, date palm has become popular in Thailand, especially cultivar Barhee, Deglet Nour, and KL1. Date palm is a dioecious plant, having separate male and female trees, and the fruit of date palm can only be collected from the female tree. Commonly, sex of date palm can be determined based on morphology of flower. However, first flowering takes 4 - 7 years. As a result, molecular techniques have become attractive because these techniques can be used to identify sex of date palm at early stage. Therefore, this research aims to develop a molecular technique called High-Resolution Melting (HRM) for gender discrimination of date palm. There are two main parts in this project, including (1) selecting suitable regions for determination date palm gender by designing primers from areas with variation between male and female tree. Then, using uMELT software to predict male and female melting profiles. (2) performing HRM analysis using the primer pairs that were designed to discriminate the gender of the date palm. The results demonstrated that two different regions of the 12th chromosome (single nucleotide polymorphisms: SNPs and genes) were found to be good candidates for gender discrimination. Both SNPs and genes contain nucleotide variation between male and female and thus good for an HRM assay. Primer pairs were designed based on both SNPs (PDK_30s1202771) and genes sequences (MYBA, BAG and TIF-2). The analyses shows that the TIF2 primers was the only one that can be used to discriminate gender of two out of three date palm cultivars (Barhee and Deglet Nour) considered from melting temperatures (Tm) of male and female date palms (cultivar Barhee Tm of male trees = 86.36 ± 0.06, Tm of female trees = 86.25 ± 0.04 and Deglet Nour Tm of male trees = 86.83 ± 0.04, Tm of female trees = 86.53 ± 0.05) and the differences of melting curve shape between male and female tree.