Causative Mutation Identification in Non-Vascular Plants
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Solution Overview
Problem
Current methods for identifying causative mutations in plants require outcrossing, which is time-consuming and costly, especially for sterile mutants, and limits pipeline throughput, as they necessitate reproductive cycles and segregation analysis.
Innovation Solution
A computer-implemented method that aligns DNA sequences of test and comparison samples to a reference sequence, filters mismatches to identify candidate mutations, bypassing the need for outcrossing by selecting samples based on complementation groups or absence of causative mutations, using a processor to execute this process.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If outcrossing is performed to reduce background mutations, then identification accuracy is improved, but time consumption increases and pipeline throughput decreases
Solution Approach 1:
The patent performs preliminary sequencing of the F1 generation mutants before outcrossing is completed. By sequencing the F1 mutants and comparing them with the F2 generation, the causative mutation can be identified early in the process, eliminating the need to wait for complete outcrossing and segregation analysis to finish.
Solution Approach 2:
The patent creates a digital copy of the genomic data through sequencing and stores it for later analysis. The F1 and F2 generation sequences are stored and compared computationally, allowing rapid identification of causative mutations without requiring physical outcrossing to completion for analysis.
2Measurement precision
If outcrossing is performed to reduce background mutations, then identification accuracy is improved, but pipeline throughput decreases
Solution Approach 1:
The patent performs preliminary sequencing and computational analysis in the F1 generation before the traditional outcrossing and segregation analysis is completed. This allows multiple mutants to be processed simultaneously and rapidly, significantly increasing the number of mutations that can be identified per unit time.
Solution Approach 2:
The patent replaces the mechanical biological process of outcrossing and segregation analysis with a computational approach. By using DNA sequencing and computer-based comparison of F1 and F2 generations, the system eliminates time-consuming physical breeding operations while maintaining identification accuracy.
3Ease of manufacture
If traditional mutation discovery pipeline is used, then identification method is simple, but sterile mutants cannot be processed
Solution Approach 1:
The patent uses digital sequencing copies of the genome rather than requiring physical outcrossing. The F1 and F2 generation DNA sequences are captured and stored as data, allowing analysis of sterile mutants without requiring them to produce offspring, thus expanding applicability to all mutant types.
Solution Approach 2:
The patent replaces the mechanical requirement of outcrossing (which fails for sterile mutants) with a molecular sequencing approach. By directly sequencing DNA from the mutant and its progeny, the method can process any mutant regardless of fertility, making the system universally applicable.
4Measurement precision
If outcrossing and segregation analysis are performed, then causative mutation identification is accurate, but cost increases
Solution Approach 1:
The patent performs sequencing and analysis in the F1 generation before completing the traditional outcrossing process. This preliminary action reduces the amount of biological material and time required, lowering costs while maintaining accuracy through early identification of causative mutations.
Solution Approach 2:
The patent replaces expensive and time-consuming physical outcrossing and segregation analysis with cost-effective DNA sequencing and computational comparison. The molecular biology approach requires fewer resources and can be scaled more efficiently than traditional breeding methods.
Data Source
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AI summary
A method for identifying a mutation associated with a phenotype of interest in a non- vascular plant, wherein the method comprises (a) aligning the DNA sequence of a reference DNA sequence and identifying a first set of sequence mismatches between the two sequences; wherein the test sample is from a mutagenized non-vascular plant; (b) aligning the DNA sequence of at least one comparison sample to the reference DNA sequence and identifying a second set of sequence mismatches between the two sequences; (c) filtering the first set of mismatches with respect to the second set of mismatches to identify a subset of mismatches that are unique to the first set of mismatches, wherein the subset of mismatches are candidate mutations for the causative mutation; wherein the test sample is from a non-vascular plant exhibiting the phenotype of interest and wherein the at least one comparison sample is from an independent non- vascular plant of the same genus that does not exhibit the phenotype of interest; and wherein the reference DNA sequence is a known reference sequence for a non-vascular plant of the genus. In addition, a method for identifying a mutation associated with a phenotype of interest in a non-vascular plant, wherein the method comprises a) aligning the DNA sequence of a reference DNA sequence and identifying a first set of sequence mismatches between the two sequences; wherein the test sample is from a mutagenized non-vascular plant; (b) aligning the DNA sequence of at least one comparison sample to the reference DNA sequence and identifying a second set of sequence mismatches between the two sequences; (c) filtering the first set of mismatches with respect to the second set of sequence mismatches to identify a subset of mismatches that are common to the first and second sets of sequence mismatches wherein the test sample and the comparison sample(s) are from independent non-vascular plants exhibiting the phenotype of interest and wherein the independent non-vascular plants are the same genus; and wherein the reference DNA sequence is a known reference sequence or a non-vascular plant of the genus or a non-vascular plant of the genus.