US 12,033,724 B2
Methods for simultaneous pooled genotyping
Justin P Gerke, Urbandale, IA (US); Heather Renee Snowgren Erickson, Ankeny, IA (US); and Frank Technow, Waterloo (CA)
Assigned to PIONEER HI-BRED INTERNATIONAL, INC., , IA (US)
Appl. No. 16/096,932
Filed by PIONEER HI-BRED INTERNATIONAL, INC., Johnston, IA (US)
PCT Filed May 3, 2017, PCT No. PCT/US2017/030754
§ 371(c)(1), (2) Date Oct. 26, 2018,
PCT Pub. No. WO2017/196597, PCT Pub. Date Nov. 16, 2017.
Claims priority of provisional application 62/335,346, filed on May 12, 2016.
Prior Publication US 2019/0116747 A1, Apr. 25, 2019
This patent is subject to a terminal disclaimer.
Int. Cl. G16B 20/40 (2019.01); C12Q 1/6827 (2018.01); C12Q 1/6895 (2018.01); G16B 20/00 (2019.01); G16B 40/00 (2019.01)
CPC G16B 20/40 (2019.02) [C12Q 1/6827 (2013.01); C12Q 1/6895 (2013.01); G16B 20/00 (2019.02); G16B 40/00 (2019.02)] 27 Claims
 
1. A method for selecting a DNA sample, comprising high density genotyping two or more progeny plants simultaneously in a pool, wherein the two or more progeny plants result from crossing a different pair of parent plants, the method comprising:
(a) collecting:
(i) genetic map distance information pertaining to one or more marker loci;
(ii) genotype information for an allele of the one or more marker loci for each parent plant of a first parental cross, wherein the first parental cross produces a first progeny plant;
(iii) genotype information for an allele of the one or more marker loci for each parent plant of a second parental cross, wherein the second parental cross produces a second progeny plant; and
(iv) pedigree information pertaining to the first progeny produced by the first parental cross and the second progeny produced by the second parental cross;
wherein the genetic map distance information is from a plant species that is the same plant species as the parent plants;
(b) providing a pooled DNA sample comprising:
a pooled genomic DNA sample comprising a first genomic deoxyribonucleotide (DNA) sample isolated from the first progeny plant and a second genomic DNA sample isolated from the second progeny plant;
(ii) a genomic DNA sample isolated from a pooled tissue sample comprising a first tissue sample from the first progeny plant and a second tissue sample from the second progeny plant; or
(iii) a genomic DNA sample isolated from an offspring produced by crossing the first progeny plant with the second progeny plant;
(c) detecting in the pooled DNA sample at least one allele of each marker locus from step (a);
(d) genotyping the pooled DNA sample for at least one marker locus detected in step (c), wherein the genotyping step comprises:
(i) building a first matrix for each marker locus detected in step (c) by calculating the probabilities that a pattern of inheritance at a previous marker locus can result in a pattern of inheritance at the marker locus based on the pedigree information and the genetic map distance information;
(ii) building a second matrix for each marker locus detected in step (c) by calculating the probabilities that an observed genotype of the pooled genomic DNA sample could be produced by each permutation of inheritance from each parent plant in the parental cross that produced the progeny plant based on each detected allele in step (c) and the pedigree information; and
(iii) determining a probability of each possible genotype for the at least one marker locus detected in step (c) in at least one progeny plant, and
(e) selecting the DNA sample based on the most probable genotype.