Non-destructive Microspore Genotyping via Tetrad Segmentation
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Solution Overview
Problem
Current methods lack the ability to non-destructively genotype microspores or pollen grains, hindering plant breeding by preventing early selection of microspores with desirable genetic compositions for further development or use in plant production.
Innovation Solution
The method involves isolating a microspore tetrad, separating it to obtain four tetrad microspores, genotyping three of them, and inferring the genotype of the fourth based on allelic segregation, allowing for the selection and potential doubling of the microspore for further growth or use in fertilization, using techniques like micromanipulation, cell sorting, or enzymatic digestion.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If traditional genotyping methods are used on microspores, then genotype information can be obtained, but the microspore is destroyed and cannot be used for further development
Solution Approach 1:
The tetrad is segmented into four individual microspores, allowing three to be used for genotyping while the fourth is preserved for development. This segmentation enables selective use of different microspores for different purposes, resolving the contradiction between obtaining genotype information and maintaining microspore viability.
Solution Approach 2:
Instead of directly genotyping the valuable fourth microspore, the patent creates copies (genotype information) from the other three microspores. This copying approach allows the original fourth microspore to remain intact and viable for further development while still obtaining the desired genotype information through the other three microspores.
2Loss of information
If all four microspores are genotyped individually, then complete genotype information is obtained, but time and resources are wasted since one microspore must be discarded
Solution Approach 1:
The patent extracts the genotype information from three microspores that are set aside for genotyping, while the fourth microspore is extracted and preserved for development. This extraction approach allows obtaining complete genotype information without genotyping all four microspores, saving time and resources.
Solution Approach 2:
The patent performs preliminary separation of the tetrad into four individual microspores before genotyping. This preliminary action allows selection of three microspores for genotyping and one for preservation, avoiding the need to genotype all four and then discard one, thereby saving time and resources.
3Measurement precision
If microspores are separated and genotyped individually, then genotype data is obtained, but the process becomes complex and time-consuming
Solution Approach 1:
The tetrad is segmented into four distinct microspores, allowing individual handling and genotyping of three while preserving the fourth. This segmentation simplifies the overall process by enabling parallel processing of genotyping and preservation tasks, reducing complexity compared to genotyping all four individually.
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
Enables non-destructive genotyping of microspores, allowing for the selection and utilization of microspores with favorable genotypes for plant breeding, facilitating the production of doubled haploid plants or specific traits, thereby enhancing plant breeding efficiency.
Implementation Method 1
The meiotic crossing over that occurs during microsporogenesis results in the segregation of alleles into four recombinant haploid products (i.e. tetrad microspores, individual microspores, or later, gametes in the pollen grains).
Data Source
Figure 1A~1D
Figure 2A~2B
Figure 2C~2D
AI summary
Methods and compositions to genotype microspores and/or pollen grains are provided. Methods provided include using meiotically-related products to non- destructively determine the genotype of one of the meiotically-related products such that it can be further used. For example, methods to obtain a genotype of a microspore are provided. The methods include: isolating tetrads, separating the tetrads to obtain four tetrad microspores; genotyping three of the four tetrad microspores, and inferring the genotype of the fourth tetrad microspore. Methods are also provided for generating doubled haploid plants from the microspores that are selected for further analysis based on inferred genotypes. Viable genotyped pollen produced by these methods, as well as seed, cells, plants, germplasm, progeny, and plant parts derived therefrom are also provided.