# US 12,601,014 B2 · Multiple KASP marker primer set for wheat plant height major genes and use thereof

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- Number: US 12,601,014 B2
- Document ID: US12601014B2
- Status: Granted
- Kind: B2
- Application number: 18/720,668
- Filed: 2023-06-28
- Published: 2025-02-13
- Granted: 2026-04-14
- Earliest claimed: 2022-08-24
- Assignees: Jiangsu Academy of Agricultural Sciences
- Applicants: Jiangsu Academy of Agricultural Sciences
- Inventors: Peng Jiang; Xu Zhang; Lei Wu; Yi He; Chang Li
- Web page: https://patentgrep.com/patent/US12601014B2

## CPC

- C12Q 1/6895: for plants, fungi or algae
- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- C12Q 1/68: involving nucleic acids
- C12Q 1/6858: Allele-specific amplification
- C12Q 2600/13: Plant traits
- C12Q 2600/156: Polymorphic or mutational markers
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

A multiplex KASP marker primer set for a set of wheat plant height major genes, consisting of a pre-primer having a nucleotide sequence as shown in SEQ ID NO. 10, a post-primer having a nucleotide sequence as shown in SEQ ID NO. 11, and a universal primer having a nucleotide sequence as shown in SEQ ID NO. 9. The multiplex KASP marker primer set achieves the simultaneous detection of the Rht-B1 and Rht-D1 genes.

## Claims

### Claim 1

A multiple primer set for Kompetitive Allele Specific PCR (KASP) assay for simultaneously detecting Rht-B1 and Rht-D1 genes controlling wheat plant height, wherein the primer set consists of a forward primer F with a nucleotide sequence consisting of SEQ ID NO. 10, a forward primer H with a nucleotide sequence consisting of SEQ ID NO. 11, and a reverse universal primer R with a nucleotide sequence consisting of SEQ ID NO. 9,
wherein the forward primer F comprises a first fluorescent label-binding sequence at its 5′ end, and targets a single nucleotide polymorphism (SNP) in the Rht-B1 gene,
wherein the forward primer H comprises a second fluorescent label-binding sequence at its 5′ end, and targets an SNP in the Rht-D1 gene,
wherein the second fluorescent label-binding sequence is different from the first fluorescent label-binding sequence,
wherein the reverse universal primer R is shared by the forward primer F and the forward primer H to enable simultaneous PCR amplification from both the Rht-B1 and Rht-D1 genes in a single reaction.

### Claim 2 (depends on 1)

A method for simultaneous detection of wheat Rht-B1 and Rht-D1 genotype, wherein the method comprises using the multiple primer set of claim 1; wherein the Rht-B1 genotype is either a Rht-B1a genotype or a Rht-B1b genotype, wherein the Rht-D1 genotype is either a Rht-D1a genotype or a Rht-D1b genotype.

### Claim 3 (depends on 2)

The method according to claim 2, wherein the method refers to PCR amplification of wheat samples using the multiple primer set, followed by fluorescence detection performed on the amplified products; if the fluorescence detection result is blue, it indicates that a genotype of the sample wheat is Rht-B1b/Rht-D1a; if the fluorescence detection result is red, it indicates that a genotype of the sample wheat is Rht-B1a/Rht-D1b; if the fluorescence detection result is green, it indicates that a genotype of the sample wheat is Rht-B1b/Rht-D1b; and if the fluorescence detection result is black, it indicates that a genotype of the sample wheat is Rht-B1a/Rht-D1a or blank.

### Claim 4 (depends on 3)

The method according to claim 3, wherein the PCR amplification refers to:
PCR reaction system: 0.07 μL of KASP Assay Mix, 2.43 μL of wheat template DNA at a concentration of 20 ng/μL, supplemented with 2×KASP Master Mix to 5 μL, wherein
each 100 μL of the KASP Assay Mix comprises: 12 μL of the primer F at a concentration of 100 μM, 12 μL of the primer H at a concentration of 100 μM, and 30 μL of the universal primer R at a concentration of 100 μM, supplemented with ddH2O to 100 μL; and
PCR reaction procedure: 94° C. for 15 minutes; 94° C. for 20 s, 61-55° C. for 1 minutes, with a decrease of 0.6° C. per cycle for a total of 10 cycles; 94° C. for 20 s, 55° C. for 1 minutes, a total of 26 cycles.