P101
TILLING for Mutation Discovery in Rice, Wheat, Lettuce, Tomato and
Soybean Using the AdvanCE™ FS96 High-Throughput Capillary
Electrophoresis System
Dayna Loeffler, Jessica Mullenberg, Aaron Holm
Arcadia Biosciences, Inc., 410 W. Harrison St., Suite 150, Seattle, WA 98119
Deepak Dibya, Jeremy Kenseth
Advanced Analytical Technologies, Inc., 2711 South Loop Dr., Suite 4150, Ames, IA 50010
ABSTRACT: TILLING (Targeting Induced Local Lesions IN Genomes)
is a high-throughput platform for uncovering SNPs (single nucleotide
polymorphisms) in genes of interest. These SNPs can be introduced
using chemical mutagens or can be uncovered by comparing different
varieties or populations of plants and animals (EcoTILLING, Comai et
al., 2004). At Arcadia Biosciences, we have mutagenized DNA
libraries in many crops including tetraploid and hexaploid wheat, rice,
lettuce, soybean, castor and tomato. Since 2002, we have discovered
more than 4,600 SNPs in multiple crops using the Li-Cor 4200 platform
for mutation detection. Here we have evaluated a new platform that is
high-throughput, robust and easily amenable as well as having a low
cost per sample analyzed. Advanced Analytical’s AdvanCE™ FS96
system has been shown to separate enzymatically cleaved DNA
fragments in various ratios of mutant to wild type DNA in a 96-well
high-throughput format in less than an hour per run. We evaluated the
ability of the AdvanCE™ FS96 system to detect over 100 different
induced mutations in 5 different crops with a variety of pooling
strategies. We found the AdvanCE™ FS96 system to be very effective
at identifying SNPs.
METHODS: Normalized DNA samples are pooled.
Pooling is determined for each species based on
the maximum amount possible that will still allow
for detection of the majority of mutations. PCR
and heteroduplex formation is performed
according to published protocols. For wheat,
homoeolog specific primers are used. Next, a
portion of PCR product is subjected to enzymatic
digestion, followed by dilution with a buffer and
subsequently electrophoresed for less than 1 hour
on AdvanCE™ FS96 system.
RESULTS:
6-fold pooling, diploid organisms
Lettuce:
2 mutations, both
homozygous
Li-Cor & FS96 COMPARISON: Tomato, 6-fold pooled heterozygous SNP
SNP: cut 1 SNP: cut 2
Full length PCR
650bp
520bp
Advanced Analytical, capillary view
Lower marker
Li-Cor
700
channel
Li-Cor
800
channel
Advanced Analytical
gel view, one capillary
Upper marker
The 700 nm and the 800 nm Li-Cor
images show 6 lanes of an assay.
The Advanced Analytical images
show one capillary, which was loaded
with PCR product that corresponds to
the lane in the Li-Cor image that
contains the highlighted SNP. The
upper and lower markers in the
Advance Analytical images are used
to normalize the capillaries.
Hexaploid wheat, 2-fold pooling
F01
F02
F03
F04
F05
F06
F07
F08
F09
F10
F11
F12
cut 1
(bp)
900
900
630
545
850
250
400
465
465
770
920
500
cut 2
(bp)
300
300
570
655
350
950
800
735
735
430
280
700
The full length PCR product for these
samples is 1200bp. Each lane has a SNP.
The sizes are listed in the chart above.
Hexaploid wheat, 4-fold pooling
Peaks annotated with
peak height
Soybean:
2 identical genes
amplified (equal to
12-fold pooling)
Rice: The same mutation, on the left heterozygous
and on the right homozygous
The image to the left shows the gel view, and the
image above the capillary view for a 4-fold pooled,
hexaploid, wheat sample with two SNPs. Cut fragments
are expected at roughly 125 &1075bp and 290 & 910bp.
The faint peak at 620bp is unexpected.
CONCLUSIONS:
The AdvanCE™ FS96 system is capable of detecting SNPs in multiple
species, with a sensitivity equal to or greater than the Li-Cor platform.
The AdvanCE™ FS96 system offers multiple advantages over the Li-Cor
4200 system, including a shorter assay time and a smaller environmental
footprint with no need for acrylamide gels or formamide.