DNA Fingerprinting of Egyptian Dry Date Palm Cultivars Using Amplified Fragment Length Polymorphism Technique

Gadalla, Ezz Eldin GadAlla; Ibrahim, Ibrahim A.; Saunders, James A.; Khattab, Magda M.

doi:10.21608/esjp.2023.370492

DNA Fingerprinting of Egyptian Dry Date Palm Cultivars Using Amplified Fragment Length Polymorphism Technique

Document Type : Scientific articles

Authors

¹ Horticulture and Biotecnology, CLDPRD, ARC

² Genetic Engineering and Biotechnology Research Institute (GEBRI), Sadat City, Minufiya University.

³ Alternative Crops and Systems Lab., Plant Sciences Institute, Beltsville Agricultural Research Center, USDA / ARS, MD.

⁴ Department of Pomology, Faculty of Agriculture, Cairo University, Giza, Egypt.

10.21608/esjp.2023.370492

Abstract

Amplified Fragment Length Polymorphism (AFLP) DNA analysis of date palm cultivars is based on a multi-step process with DNA digestion, fragment ligation to adapters, pre-selective PCR, selective PCR amplification and fragment separation. DNA analysis of date palm cultivars Sakkoty, Malakaby, Gondeila, Shamia and Bartamuda with a single PCR primer set based on the EcoRI and MseI cut site plus three selective nucleotides from each end of the DNA template. MseI is a 4-bp frequent cutting endonuclease which is typically mixed together with the less frequent 6-bp cutting endonuclease known as EcoRI. The use of these two restriction enzymes typically digests dry date palm cultivars genomic DNA into fragments with a size range of 56 –390 bp. DNA was isolated from frozen date palm tissues . About 50 mg leaf tissue placed in a capped tube sandwiched between 500 mg of garnet and two ceramic beads. Modified lysis buffer (400 ml) was added from the DNeasy plant system. The plant tissue was completely macerated using a fast prep 120 (Q-Biogen) shaker/ basher at oscillation speed of 5.0 rpm for 40 seconds. Further DNA clean up followed manufactures instructions for DNA isolation protocol used. The PicoGreen DNA quantitation kit was used to determine DNA content in the final preparation using fluorescence measurement from a Fluoroskan Ascent microplate reader equipped with 485 /538 exitation/ emission filter settings. The strength of AFLP technique lies in the fact that multiple primers can be run from the pre-selective amplification mixture to determine if they can distinguish between unknown dry date palm samples. Six primer pairs were used to detect differences in AFLP DNA fragments patterns for dry date palm cultivars and we found three (1- E-AAC / M- CAC .2- E-ACA / M-CAA. 3- E-ACT / M-CAT. ) of them is the better to distinguish amongst date palm cultivars. The dendogram produced by the Jaccard measure cluster analysis of the pooled AFLP data from three primers pairs showed that date palm dry cultivars are arranged according to DNA fingerprinting as following: Sakkoty, Malakaby, Gondeila, Shamia and Bartamuda. It means that Sakkoty and Malakaby are very closed, Shamia and Bartamuda are very closed too, while Gondeila is in between two cluster cultivars but more related to that involved Sakkoty and Malakaby . The use of DNA cultivar typing in dry group samples are useful for cultivar identification and breeding programs.

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