Transcript document

Development of TaqMan PCR Zygosity Assay to Detect the Maize bm3 Mutant
N. VanOpdorp, W. Chen, M. Avery, T. King, C. Channabasavaradhya, and S. Kumpatla
Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268
Abstract
Materials and Methods
Plant Materials:
Corn leaf samples containing homozygous bm3 alleles and
wild type COMT alleles were collected from a diversity of
heterotic groups which included the lancaster, stiff stalk, and
iodent backgrounds. Corn leaf samples from bm3 segregating
populations were also obtained to validate the TaqMan PCR
analysis tool.
Methods:
Corn genomic DNA fragments of a partial COMT gene were
amplified from twelve bm3 lines and three non-bm3 lines. PCR
products were visualized on a 2% E-Gel (Invitrogen, Carlsbad,
CA) and then extracted on 0.8% E-Gel CloneWells. Purified
PCR products were then cloned into pCR4-TOPO vector
(invitrogen, Carlsbad, CA) per manufacturer’s instruction.
Based on the DNA consensus sequences from the bm3 lines,
primers (BM3_F and BM3_R) and probe (BM3_Probe) specific
to the junction where partial sequences from COMT gene at
the 3’ end was deleted for identifying the mutant allele; primers
(COMT_F and COMT_R) and probe (COMT_probe) within the
deleted region for identifying the wild type alleles were
designed. Primer Express 3.0 was used for TaqMan assay
design.
BM3 deletion
A
BM3_F
BM3_R
COMT_R COMT_Probe COMT_F
BM3
deletion
B
bmr
Fig. 1 BMR corn plant next to a non-BMR corn plant
BM3_F
BM3_Probe
specific to bm3 mutation and intact COMT gene. (A) COMT
gene with dotted lines represents bm3 deletion. COMT gene
specific primers are within the deletion site. (B) bm3 mutant
gene..
Wild type COMT
Results and Discussion
Cloning, Assay Design, and Validation of TaqMan Analysis:
Precise sequence information was needed to design a gene
specific TaQman assay for bm3. Public information was used to
design 2 oligos which amplified the partial COMT gene from the
bm3 and non bm3 lines, and then these fragments were then
cloned and sent to Cogenics to obtain the full length sequence of
~1800 bases. The bm3 lines tested all contain a large deletion
mutation at the 3’ exon of the COMT gene and this mutation was
used for the gene specific TaqMan assay design. Real time
PCR was used to test the efficiency of the assay. FAM was
used to monitor the amplicon of the bm3 and VIC for the wild
type COMT and correct genotype calls were made based on the
allelic discrimination between the FAM and VIC reporter dyes.
The real time PCR results were then validated using end-point
TaqMan PCR which would allow the assay to be run on any
regular PCR machine fit for 96 or 384 wells which read FAM and
VIC. Samples from bm3 segregating populations were run
through the end-point TaqMan PCR assay and genotype results
matched 100% with phenotype data from the field.
BM3_R
Fig. 2: Schematic presentation of COMT gene with primers
RFU (VIC)
Brown midrib (BMR) corn varieties have a natural mutation that
results in low lignin in their cell walls, leading to increased
digestibility, which is a highly desirable trait for silage maize
production. Based on the sequence information from the
public database, we designed oligos to amplify the
COMT(bm3, wild type) gene from twelve diverse maize bm3
inbred lines and three non bm3 maize inbred lines. The PCR
products of the bm3 lines and non bm3 lines were cloned and
sequenced. An endpoint TaqMan PCR based zygosity assay
was then developed to specifically detect and to test the
zygosity status at the bm3 locus. The assay utilizes a biplex of
oligonucleotides specific to the bm3 deletion at the 3’ end of
the exon and to the corresponding wild type sequences in the
same assay. Zygosity is determined by the presence/absence
of the bm3 mutant and the wild type COMT alleles. This high
throughput PCR based molecular characterization of bm3
mutants will greatly enhance the breeding process for BMR
maize lines.
Hemizyg. bm3
Neg. Control
Homozyg bm3
RFU (FAM)
Fig.3 bm3 zygosity determination with end-point TaqMan
assay using KLIMs. A graph with RFU (relative fluorescence
unit) of FAM as x-axis and VIC as y-axis was generated.
Zygosity calls were made based on the cluster separation in a
cluster view.
Conclusions
A gene specific end-point TaqMan PCR assay for bm3
zygosity analysis has been developed and validated.
Zygosity is determined by the presence/absence of the
bm3 mutant and the wild type COMT alleles.
The use of this assay will be highly valuable in breeding
with the bm3 gene especially when stacking other BMR
genes in the same population.
Dow AgroSciences Confidential