Methods for Extraction and Amplification of DNA from Soybean Seed

Extraction and amplification of plant DNA is an integral part of using molecular technology for crop improvement such as in molecular marker-assisted selection (MAS). In soybean [Glycine max (L.) Merr.], leaves are generally the tissue of choice for DNA extraction. A comparison was made between usin...

Full description

Saved in:
Bibliographic Details
Published in:Seed technology 2005-01, Vol.27 (1), p.89-94
Main Authors: Bolton, Melvin D., Nelson, Berlin D., Sparks, Robert B., Sparks, Robert D., Santoso, Adi
Format: Article
Language:English
Subjects:
Annealing
Cyst nematodes
DNA
Gels
Leaves
Microsatellite repeats
Polymerase chain reaction
Seeds
Soybeans
Water purification
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Extraction and amplification of plant DNA is an integral part of using molecular technology for crop improvement such as in molecular marker-assisted selection (MAS). In soybean [Glycine max (L.) Merr.], leaves are generally the tissue of choice for DNA extraction. A comparison was made between using standard leaf extracted DNA and seed extracted DNA while utilizing a marker-assisted selection protocol to detect resistance to Heterodera glycines Ichinohe, the soybean cyst nematode (SCN) and Phytophthora sojae Kaufm. & Gerd., the cause of Phytophthora root rot. The protocol was unsuccessful using seeds, thus experiments were conducted to determine modifications that would allow seeds as the DNA source. The resulting procedure consisted of extracting DNA from seed radicles and using a disk-based DNA purification system. The DNA was amplified with polymerase chain reaction (PCR) using microsatellite primers Satt309 and Sat_168 to detect the rhg 1 SCN resistance gene and Satt 159 and Satt 152 to detect the Rps 1 Phytophthora resistance gene. The resulting product was diluted 1:100 in TE buffer and 1 μl aliquot was reamplified and then electrophoresed in an agarose gel. A second protocol was developed by changing PCR conditions of the standard leaf DNA protocol to use undiluted DNA extract. These protocols from DNA extraction to gel completion are easy to conduct and can be completed in about 7 to 9 hours. Both protocols resulted in consistent visualization of microsatellite markers for resistance to both H. glycines and P. sojae and were equal to the results using leaf tissue as the DNA source. Using seeds instead of leaves as the DNA source can simplify MAS protocols.
ISSN:1096-0724