Method for detecting molecular markers based on KASP technology

A technology of molecular markers and technical detection, which is applied in the direction of biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as complex process, long time-consuming, error-prone, etc.

Pending Publication Date: 2020-10-27
CROP RES INST SHANDONG ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Gel electrophoresis detection generally requires multiple steps such as gel preparation, spottin...

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  • Method for detecting molecular markers based on KASP technology
  • Method for detecting molecular markers based on KASP technology

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Experimental program
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Effect test

Embodiment 1

[0029] Wheat waxy gene has been reported Wx-A1 位于7A染色体,其分子标记MAG264为共显性STS标记,在野生型264A1a的扩增片段如SEQ No.3所示;具体序列为:CGGCGGTGCCTCTCCATGGTGGTGCGCGCCACGGGCAGCGGCGGCATGAACCTCGTGTTCGTCGGCGCCGAGATGGCGCCCTGGAGCAAGACTGGCGGCCTCGGCGACGTCCTCGGGGGCCTCCCCGCCGCCATGGCCGTAAGCTTGCGCCACTGCCTTCTTATAAATGTTTCTTCCTGCAGCCATGCCTGCCGTTACAACGGGTGCCGTGTCCGTGCAGGCCAACGGTCACCGGGTCATGGTCATCTCCCCGCGCTACGACCAGTACAAGGACGCCTGGGACA

[0030] 在突变体264A1b的扩增片段如SEQ No.4所示;具体序列为:CGGCGGTGCCTCTCCATGGTGGTGCGCGCCACGGGCAGCGGCGGCATGAACCTCGTGTTCGTCGGCGCCGAGATGGCGCCCTGGAGCAAGACTGGCGGCCTCGGCGACGTCCTCGGGGGCCTCCCCGCCGCGGACGCCTTCTTATAAATGTTTCTTCCTGCAGCCATGCCTGCCGTTACAACGGGTGCCGTGTCCGTGCAGGCCAACGGTCACCGGGTCATGGTCATCTCCCCGCGCTACGACCAGTACAAGGACGCCTGGGACA

[0031] Using the 264A1a sequence to blast the wheat reference genome on the URGI website, a highly similar sequence was found at 7D and 4A.

[0032] After downloading similar sequences, DNAMAN software was used for multiple sequence alignment, and it was determined that the specific SN...

Embodiment 2

[0041] Wheat stem rust resistance gene has been reported Sr2 Located on chromosome 3B, the CAPS molecular marker csSr2 is dominant in most wheat materials in my country, similar to the molecular marker SCAR. The csSr2 amplified fragment DNA sequence is shown in SEQ No.8; specifically:

[0042] CAAGGGTTGCTAGGATTGGAAAACAAAATTGTATTCTTTGTGATTGTAATTGTTTGGTTCAAACATGAATAAAATAATATTCGATTTTCCATTGCTCTACACAACAAGTGTTCATGATTCTAGAACCCTCAAATGGTCGAGCACAAGCTCTAATTTCTTTGGAATCATGAGTGTTGGATGTCTCGCACACCTAGCTTAATACATGCAGATACTTGTTGGGAAGTAACGCTGCTAAAGAAACTTATGTATGGTTCTTAGCTAAATTAGGTTTGAGAGGGGCTAATGGTTCTATGTTGTGTAGCTTGCCCAGAAAAATGTAAGATCATAAGAGTTATCT

[0043] Compare it with the reference genome by BLAST, find multiple high-similarity sequences on the 3D and 3B chromosomes, and use DNAMAN for visual comparison after downloading. Design a site-specific primer Sr2H3 at a known SNP site, as shown in SEQ No.9 (5'-CGAGCACAAGCTCTAATTTCTTTGG AATCA-3'), the Tm value is 59 degrees, another SNP site is found at...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a molecular marker detection method, and particularly relates to a method for detecting molecular markers based ona KASP technology. The molecular markers are non-SNP and InDels. The method comprises the steps that KASP primers are designed in marker amplification target DNA sequences; for a co-dominant marker, three primers need to be designed for two target DNA sequences, and the three primers comprise two site specific primers and one common primer; for a dominant marker, two primers need to be designed for only one target DNA sequence, and the two primers comprise one site specific primer and one common primer; and the 3' end of each site specific primer is an SNP site, the Tm value of the primers is1 degree or above higher than that of a KASP joint sequence, and the size of an amplification fragment is 170 +/-130bp. According to the method, the detection range of the KASP technology is widened,and STS or SCAR is quickly detected.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a molecular marker detection method, in particular to a method for detecting molecular markers based on KASP technology. Background technique [0002] KASP (Kompetitive Allele-Specific PCR, competitive allele-specific PCR) is based on the specific matching of primer terminal bases to SNP (single nucleotide polymorphism, single nucleotide polymorphism) typing and detection of InDels (Insertions and Deletions, insertions and deletions). This technology can accurately judge SNPs or InDels widely present in genomic DNA, and is a high-throughput, low-cost, and low-error SNP typing technology. Moreover, KASP labeling can avoid laboratory gel electrophoresis, realize automation and platform operation, and have the characteristics of high throughput, low cost, and good genetic stability. In view of this feature, KASP markers have played an important role in wheat gene mapping, map-based cl...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858
CPCC12Q1/6858C12Q2531/113C12Q2563/107
Inventor 李玮宋国琦李根英李吉虎李玉莲张淑娟张荣志高洁
Owner CROP RES INST SHANDONG ACAD OF AGRI SCI
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