TILLING to detect induced mutations in soybean.
BACKGROUND
Soybean (Glycine max L. Merr.) is an important nitrogen-fixing crop that provides much of the world's protein and oil. However, the available tools for investigation of soybean gene function are limited. Nevertheless, chemical mutagenesis can be applied to soybean followed by screening for mutations in a target of interest using a strategy known as Targeting Induced Local Lesions IN Genomes (TILLING). We have applied TILLING to four mutagenized soybean populations, three of which were treated with ethyl methanesulfonate (EMS) and one with N-nitroso-N-methylurea (NMU).


RESULTS
We screened seven targets in each population and discovered a total of 116 induced mutations. The NMU-treated population and one EMS mutagenized population had similar mutation density (approximately 1/140 kb), while another EMS population had a mutation density of approximately 1/250 kb. The remaining population had a mutation density of approximately 1/550 kb. Because of soybean's polyploid history, PCR amplification of multiple targets could impede mutation discovery. Indeed, one set of primers tested in this study amplified more than a single target and produced low quality data. To address this problem, we removed an extraneous target by pretreating genomic DNA with a restriction enzyme. Digestion of the template eliminated amplification of the extraneous target and allowed the identification of four additional mutant alleles compared to untreated template.


CONCLUSIONS
The development of four independent populations with considerable mutation density, together with an additional method for screening closely related targets, indicates that soybean is a suitable organism for high-throughput mutation discovery even with its extensively duplicated genome.
Cooper
Jennifer L
JL
Fred Hutchinson Cancer Research Center, Seattle, WA 98107, USA. jlcooper@fhcrc.org
Till
Bradley J
BJ
Laport
Robert G
RG
Darlow
Margaret C
MC
Kleffner
Justin M
JM
Jamai
Aziz
A
El-Mellouki
Tarik
T
Liu
Shiming
S
Ritchie
Rae
R
Nielsen
Niels
N
Bilyeu
Kristin D
KD
Meksem
Khalid
K
Comai
Luca
L
Henikoff
Steven
S
D001483:Base Sequence; D017931:DNA Primers; D018744:DNA, Plant / Q000235:genetics; D005020:Ethyl Methanesulfonate / Q000494:pharmacology; D008770:Methylnitrosourea / Q000494:pharmacology; D016296:Mutagenesis; D013025:Glycine max / Q000235:genetics*
D017931:DNA Primers; D018744:DNA, Plant; D008770:Methylnitrosourea; D005020:Ethyl Methanesulfonate