Patent ID: 11884972
Assignee: CARLSBERG A/S
Field: Biotechnology (Chemistry)
Classification: CPC C  A | IPC A  C

Claim 19:
20. A method of identifying a sub-pool of plants of a predefined plant species or reproductive parts thereof, wherein the sub-pool comprises a plant carrying one or more predetermined mutations in one or more nucleotides of interest (NOI) in a target sequence, wherein the predetermined mutation is a deletion or substitution of one or more NOIs, said method comprising the steps of:
a) Providing a plurality of reproductive parts of the plant species, the plurality comprising at least 100 reproductive parts with different genotypes;
b) Subjecting said reproductive parts to random mutagenesis, thereby obtaining reproductive parts of generation M0;
c) Growing said reproductive parts of generation M0 into mature plants and obtaining reproductive parts from said mature plants, wherein said reproductive parts are reproductive parts of generation M1;
d) Obtaining reproductive parts of generation M1 from said mature plants, thereby obtaining a pool of reproductive parts;
e) Dividing said pool of reproductive parts of generation M1 into sub-pools, wherein all reproductive parts from a given mature M0 plant are placed into the same sub-pool, wherein each sub-pool comprises a plurality of genotypes;
f) Preparing genomic DNA (gDNA) samples, each sample comprising gDNA from each genotype within a sub-pool, while maintaining the potential for multiplication of plants of each genotype within said sub-pool;
g) Performing a plurality of PCR amplifications, each comprising a gDNA sample from one sub-pool, one or more sets of primers, wherein each set of primers flanks a target sequence, and PCR reagents, thereby amplifying one or more target sequences;
h) Detecting one or more PCR amplification products comprising one or more target sequences, said one or more target sequences comprising the one or more mutations in the one or more NOI, thereby identifying one or more sub-pools comprising said one or more mutations,
wherein the pool obtained in step (d) comprises at least 100,000 reproductive parts; or
wherein each sub-pool obtained in step (e) comprises at least 100 reproductive parts with different genotypes.