Floury 2 gene-specific assay in maize for floury (fl2) trait introgression
View Patent ↗The disclosure concerns compositions and methods for determining the zygosity of corn plants containing one or more floury2 (fl2) mutations. In embodiments, the disclosure concerns a gene specific PCR-based molecular (KASPar®) assay for identifying the floury2 trait in plants. In certain embodiments, compositions and methods are disclosed for determining the zygosity of corn plants with respect to the fl2 allele. In particular embodiments, the assay may be used for fl2 germplasm identification, accelerating introgression and molecular breeding programs in corn and other plants.
1. A method for determining the zygosity and/or presence/absence of an allele in a corn plant tissue, the method comprising:
obtaining a sample of isolated genomic DNA from the corn plant tissue;
contacting the isolated genomic DNA with at least one nucleic acid molecule comprising a nucleotide sequence capable of hybridizing to SEQ ID NO:9 under high stringency conditions and at least one nucleic acid molecule capable of hybridizing to SEQ ID NO:10 under high stringency conditions; and
determining the zygosity of fl2 mutation in the isolated genomic DNA,
wherein the fl2 mutation comprises a (C/T) single nucleotide polymorphism (SNP) at nucleotide position 495 of SEQ ID NO:9 or SEQ ID NO:10,
specifically detecting the (C/T) SNP at nucleotide position 495 of SEQ ID NO:9 or SEQ ID NO:10 via a KASPar® PCR assay,
generating zygosity genotype data of the fl2 mutation, and
comparing the zygosity genotype data with phenotype data collected from the field,
wherein the accuracy rate of homozygous genotype data with the phenotype data collected from the field is at least about 97% and the accuracy rate of heterozygous genotype data with the phenotype data collected from the field is at least about 42%.
2. The method of claim 1 , further comprising:
contacting the isolated genomic DNA with two nucleic acid molecules each comprising a nucleotide sequence capable of hybridizing to SEQ ID NO:9 under high stringency conditions, and two nucleic acid molecules capable of hybridizing to SEQ ID NO:10 under high stringency conditions;
amplifying the nucleotide sequence of the isolated genomic DNA between nucleotide sequences of the isolated genomic DNA that hybridize to the two nucleic acid molecules each comprising a nucleotide sequence capable of hybridizing to SEQ ID NO:9 under high stringency conditions;
amplifying the nucleotide sequence of the isolated genomic DNA between nucleotide sequences of the isolated genomic DNA that hybridize to the two nucleic acid molecules each comprising a nucleotide sequence capable of hybridizing to SEQ ID NO:10 under high stringency conditions;
including in the amplifying reaction at least one nucleic acid molecule comprising a nucleotide sequence capable of hybridizing to SEQ ID NO:9 under high stringency conditions that is labeled with a first reporter, and at least one nucleic acid molecule capable of hybridizing to SEQ ID NO:10 under high stringency conditions that is labeled with a second reporter; and
detecting the levels of the first and second reporter.
3. The method of claim 2 , wherein the first reporter and the second reporter are fluorescent dyes with distinguishable excitation/emission spectra.
4. The method of claim 3 , wherein the first reporter is FAM, and the second reporter is VIC.
5. The method of claim 1 , wherein the at least one nucleic acid molecule comprising a nucleotide sequence capable of hybridizing to SEQ ID NO:9 is between 10 and 35 nucleotides in length.
6. The method of claim 5 , wherein the at least one nucleic acid molecule comprising a nucleotide sequence capable of hybridizing to SEQ ID NO:9 is between 15 and 30 nucleotides in length.
7. The method of claim 5 , wherein one of the at least one nucleic acid molecules comprising a nucleotide sequence capable of hybridizing to SEQ ID NO:9 is at least 95% identical to between 10 and 35 contiguous nucleotides of SEQ ID NO:9.
8. The method of claim 7 , wherein the at least one nucleic acid molecule comprising a nucleotide sequence capable of hybridizing to SEQ ID NO:9 is selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.
9. The method of claim 1 , wherein at least one nucleic acid molecule comprising a nucleotide sequence capable of hybridizing to SEQ ID NO:9 under high stringency conditions is labeled with a fluorescent dye, and at least one nucleic acid molecule capable of hybridizing to SEQ ID NO:10 under high stringency conditions is labeled with a second fluorescent dye with a distinguishable excitation/emission spectra.
10. The method of claim 9 , wherein at least one nucleic acid molecule comprising a nucleotide sequence capable of hybridizing to SEQ ID NO:9 under high stringency conditions is labeled with FAM, and at least one nucleic acid molecule capable of hybridizing to SEQ ID NO:10 under high stringency conditions is labeled with VIC.
11. The method of claim 2 , wherein amplifying the nucleotide sequence comprises amplifying in a high throughput PCR reaction.
12. A method for reliably and predictably introgressing a trait for high lysine content into plant germplasm, said method comprising:
crossing a plant having a mutation in the fl2 gene with another plant;
obtaining a sample of isolated genomic DNA from a progeny plant produced by the cross;
contacting the isolated nucleic acid with at least one nucleic acid molecule having a nucleotide sequence capable of hybridizing to SEQ ID NO:10 under high stringency conditions;
selecting progeny from the cross that comprise a mutation in the fl2 gene,
wherein the mutation in the fl2 gene is associated with a trait for high lysine content and comprises a (C/T) single nucleotide polymorphism (SNP) at nucleotide position 495 of SEQ ID NO:10,
reproducing a plant from which a sample was obtained that binds the at least one nucleic acid molecules with high stringency, and
producing a genetically engineered plant wherein the trait for high lysine content has been introgressed into the germplasm of the genetically engineered plant via a KASPar® PCR assay, and
detecting the allele-specific genotype of the fl2 mutation comprised by the genetically engineered plant, and
comparing allele-specific genotype data to high lysine phenotype data collected from the field,
wherein the accuracy rate of homozygous allele-specific genotype data with the phenotype data collected from the field is at least about 97% and the accuracy rate of heterozygous allele-specific genotype data with the phenotype data collected from the field is at least about 42%.
13. The method of claim 12 , wherein the at least one nucleic acid molecule comprising a nucleotide sequence capable of hybridizing to SEQ ID NO:10 is between 10 and 35 nucleotides in length.
14. The method of claim 12 , wherein the at least one nucleic acid molecule comprising a nucleotide sequence capable of hybridizing to SEQ ID NO:10 is between 15 and 30 nucleotides in length.
15. The method of claim 12 , wherein the at least one nucleic acid molecule capable of hybridizing to SEQ ID NO:10 under high stringency conditions is labeled with a fluorescent dye with a distinguishable excitation/emission spectra.
16. The method of claim 15 , wherein the fluorescent dye with a distinguishable excitation/emission spectra is VIC.
17. The method of claim 15 , wherein the fluorescent dye with a distinguishable excitation/emission spectra is FAM.