Systems and methods for pericarp genotyping
a technology of pericarp and genotyping, applied in the field of systems and methods for pericarp genotyping, can solve the problems of pericarp separation and removal, chemical requirements, time-consuming and laborious, etc., and achieve the effect of sufficient dna yield, sufficient dna yield, and sufficient dna yield
Active Publication Date: 2016-03-03
PIONEER HI BRED INT INC
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Benefits of technology
The patent describes a method for isolating pure maternal DNA from seeds by collecting maternal seed tissue, washing it, and then dissociating and homogenizing it to obtain a solution. The solution is then centrifuged to obtain a supernatant, and a molecular analysis is performed on the supernatant DNA. This method can be used to determine the maternal lineage of seeds by analyzing the DNA of the maternal seed tissue. The technique involves collecting the maternal seed tissue, washing it with a solution containing sodium dodecyl sulfate, dissociating and homogenizing it using a cell dissociator, extracting DNA from the cells, and performing a molecular analysis of the extracted DNA. The method can also involve using whole genome amplification to obtain sufficient DNA yield. The molecular analysis can be genotyping, which allows for the identification of the maternal lineage of seeds from multiple seeds.
Problems solved by technology
This, coupled with the relatively small size of most seeds, can make separation and removal of pericarp difficult, or at least time consuming and laborious.
However, such processes require chemicals, as well as costs and handling associated with them.
Most importantly, the chemicals used combined with the significant amount of time of soaking may damage or otherwise affect the DNA in the pericarp.
Water soaking is not as effective as chemicals at loosening the pericarp.
Furthermore, it has been difficult, if not impossible, to completely remove all interior seed tissue, such as endospermic tissue, from the pericarp with the water soaking method, which makes it difficult to achieve the purity level needed or desired for accurate molecular analysis.
In addition, the pooled DNA from multiple seeds, particularly from commercial seeds, brings a potential risk of contamination due to the mixing of seed source.
Method used
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Pericarp Genotyping
[0030]A. Pericarp Peeling
[0031]Kernels of corn were removed from the cob and soaked for 60 minutes in deionized water. A scalpel blade was sterilized using a bead sterilizer. The scalpel was used to cut the back side of the seeds (away from the embryo) near the tips, as shown in FIG. 1a. The scalpel was again sterilized using a bead sterilizer and cooled in sterile water. The scalpel was then used to cut along the outer edge of the kernel, as shown in FIG. 1b. Forceps were sterilized in a bead sterilizer, cooled, then used to peel the pericarp from the kernel, as shown in FIG. 1c. The pericarp tissue from each kernel was then placed in microcentrifuge tubes.
[0032]B. Pericarp Washing
[0033]Three different washing solutions were tested. The best results were achieved washing with 1% sodium dodecyl sulfate (SDS) solution, although adequate results were achievable using water and ethanol. An alternative washing method using sonication also gave adequate results. The wa...
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The invention includes methods for obtaining samples of maternal tissue from seeds, obtaining genetic material from the maternal seed tissue, and performing a molecular analysis on the genetic material from the maternal seed tissue to determine maternal lineage of a single seed. The invention also includes methods for establishing a consensus maternal genotype from maternal seed tissues obtained from multiple seeds as well as methods for determining paternal lineage.
Description
BACKGROUND OF THE INVENTION[0001]A seed is an embryonic plant enclosed in a protective outer covering called the seed coat. In monocots, the seed coat is fused with the pericarp, which is composed of multiple layers of cells and contains genetic code from the maternal parent only. As such, pericarp tissue can be used to identify the maternal lineage of a seed. Paternal lineage can be further derived from the pericarp genotype in combination with the seed (embryo or endosperm) genotype, which contains the genetic information from both maternal and paternal background. Determining lineage is important for germplasm security to ensure that proprietary parental lines remain proprietary.[0002]Tissues or other adhering structures connect the pericarp to the interior part of the seed, mainly endospermic tissue. This, coupled with the relatively small size of most seeds, can make separation and removal of pericarp difficult, or at least time consuming and laborious. Molecular analysis of im...
Claims
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IPC IPC(8): C12Q1/68
CPCC12Q1/6895C12Q2600/13C12Q2600/16C12Q1/6806C12Q2600/156C12Q2527/125
Inventor SCHARES, JUSTINYUN, YUEZHAO, CHENGFENG
Owner PIONEER HI BRED INT INC
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