Development of TaqMan PCR Zygosity Assay to Detect the Maize bm3 Mutant

1. 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 Wild type COMT Hemizyg. bm3 Neg. Control Homozyg bm3 RFU (FAM) bmr Abstract 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. BM3deletion A BM3_F BM3_R COMT_R COMT_Probe COMT_F BM3 deletion B BM3_F BM3_Probe BM3_R Fig. 2:Schematic presentation of COMT gene with primers 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.. Fig. 1 BMR corn plant next to a non-BMR corn plant 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. 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. Fig.3bm3 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