TaqMan Assay for Maize bm3 Zygosity Detection

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Solution Overview

Problem

Current methods lack a rapid and efficient way to determine the zygosity of plants containing brown midrib 3 (bm3) mutations, which are crucial for enhancing the breeding process of BMR-containing plant lines due to their reduced lignin content and improved digestibility.

Innovation Solution

A high-throughput PCR-based molecular characterization method using endpoint hydrolysis probe PCR assays, specifically a TaqMan assay, is developed to detect and test the zygosity status at the bm3 locus by utilizing oligonucleotides specific to both bm3 mutant and wild-type COMT alleles, allowing for accurate determination of plant zygosity.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If traditional breeding methods are used to determine zygosity of bm3 mutations, then the breeding process can identify plants with desired traits, but the process is time-consuming and lacks efficiency

Engineering Contradiction:

Improvebreeding process efficiencyVSAvoidtime to determine zygosity

Core Design Contradiction:

ProductivityVSLoss of time

Solution Approach 1:

The patent replaces traditional mechanical breeding evaluation methods with a molecular biology-based PCR assay system. Specifically, it uses endpoint hydrolysis probe PCR (TaqMan assay) to detect and determine zygosity at the bm3 locus, substituting phenotypic observation and manual breeding tracking with automated molecular detection that provides rapid, accurate genotyping results.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

2Measurement precision

If molecular detection methods are developed for bm3 locus, then zygosity determination becomes rapid and accurate, but the method complexity and development requirements increase

Engineering Contradiction:

Improvezygosity determination accuracyVSAvoidmolecular assay complexity

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The patent introduces specific molecular intermediaries - oligonucleotide probes and primers designed to bind to the bm3 locus - as mediators between the target DNA and the detection system. These intermediaries enable specific recognition and amplification of the bm3 mutation sequences, allowing accurate zygosity determination through standardized PCR technology while managing the complexity of molecular detection.

Inventive Principle:

Principle #24Intermediary (Mediator)

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

This method enhances the breeding process by providing a rapid and reliable means to identify and select for plants with desired low lignin content traits, improving the efficiency of introgressing the bm3 allele into corn germplasm, thereby increasing fiber digestibility and animal feed quality.

Implementation Method 1

Methodology Applied

Scientific EffectPolymerase Chain Reaction (PCR):

Implementation Method 2

endpoint hydrolysis probe PCR-based zygosity assay (which is referenced as a TaqMan assay herein)

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Data Source

PatentUS9222141B2Use of brown midrib-3 gene specific markers in maize for trait introgression

Publication Date: 2015.12.29 AGRIGENETICS INC

AI summary

This disclosure concerns compositions and methods for determining the zygosity of corn plants containing one or more brown midrib (BMR) mutations. The disclosure also concerns methods that are useful for enhancing the breeding process for BMR corn. In certain embodiments, compositions and methods for determining the zygosity of corn plants with respect to the bm3 allele.