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KASP molecular marker for high-throughput detection of high oleic acid mutation site of cotton GhFAD2-1 gene

A technology of molecular markers and mutation sites, which is applied in the direction of DNA/RNA fragments, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of long detection cycle, low detection efficiency, high detection cost, etc., and achieve long detection cycle, Low throughput, high cost effect

Pending Publication Date: 2021-12-07
VEGETABLE RES INST OF SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, using Sanger sequencing method to detect hybrid offspring has low detection efficiency, long detection cycle, and high detection cost. High-throughput detection methods are urgently needed in cotton high-oleic acid breeding and production.

Method used

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  • KASP molecular marker for high-throughput detection of high oleic acid mutation site of cotton GhFAD2-1 gene
  • KASP molecular marker for high-throughput detection of high oleic acid mutation site of cotton GhFAD2-1 gene
  • KASP molecular marker for high-throughput detection of high oleic acid mutation site of cotton GhFAD2-1 gene

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Example 1: Development of closely linked KASP markers for cotton high oleic trait genes GhFAD2-1A and GhFAD2-1D

[0029] 1. KASP primer design

[0030] Extract the 100 bp flanking sequences around the 429nt site of the GhFAD2-1A / D gene coding region, use Primer5.0 software to design 3 sets of KASP primers, and use the ArrayTape platform of Douglas Scientific Company to detect the amplification effects of KASP-A429 and KASP-D429 And the polymorphism is the best, and the InDel difference between GhFAD2-1A and GhFAD2-1D in wild type and high oleic acid mutant can be clearly distinguished.

[0031] The primers for the molecular marker KASP-A429 are:

[0032] 1) Two specific primers:

[0033] GhFAD2-1A-HEX: 5'-GAAAATCAGTCACCGCCGTCAt-3', as shown in SEQ ID No.3;

[0034] GhFAD2-1A-R: 5'-ACTCGACCGGGTGGATTGTTT-3', as shown in SEQ ID No.4;

[0035] 2) A universal primer:

[0036] GhFAD2-1AD-FAM: 5'-GAAAATCAGTCACCGCCGTCAC-3', as shown in SEQ ID No.5;

[0037] The primers fo...

Embodiment 2

[0054] Example 2: Application of KASP markers

[0055] In order to verify the practicability of the KASP marker of the present invention, the cotton materials described in Table 1 were used for detection and analysis. Among them, H01-H92 is Lumian 418 as the female parent and high oleic acid cotton mutant m1-2 (Chen Yizhen, Fu Mingchuan, LiHao, et al., High-oleic acid content, nontransgenic allotetraploid cotton (Gossypium hirsutum L.) generated by knockout of GhFAD2 genes with CRISPR / Cas9system.Plant Biotechnology Journal, 2021,19:424-426.) The F2 segregation population prepared for male parent crosses, H93 and H94 are high oleic acid cotton mutant m1-2, used as Positive control, H95 and H96 are Lumian 418, used as negative control.

[0056] Table 1 Phenotype and genotype information of cotton segregation population KASP primer list

[0057]

[0058]

[0059] The results are shown in Table 1 and figure 1 As shown, after KASP analysis, among the 92 individual plants o...

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Abstract

The invention discloses a KASP molecular marker for high-throughput detection of a high oleic acid mutation site of a cotton GhFAD2-1 gene. The molecular marker linked with a GhFAD2-1A nucleotide mutation site is KASP-A429, and the molecular marker linked with a GhFAD2-1D nucleotide mutation site is KASP-D429. The invention further designs specific amplification primers and universal primers of the GhFAD2-1A gene and the GhFAD2-1D gene to perform PCR amplification on a cotton sample to be detected, then an LGC high-throughput marker detection platform is applied to detect allelic variation of the GhFAD2-1A gene and the GhFAD2-1D gene of the cotton, and if it is detected that the GhFAD2-1A gene and the GhFAD2-1D gene in the cotton sample are in homozygous mutation at the same time, the cotton sample is a high oleic acid cotton sample. According to the method, large group screening in the high oleic acid breeding process of cotton becomes more efficient, economical, simple and accurate.

Description

technical field [0001] The invention relates to a KASP molecular marker for high-throughput detection of the high oleic acid mutation site of cotton GhFAD2-1 gene, which belongs to the field of biotechnology. Background technique [0002] Cotton is an important fiber and oil crop. Fiber accounts for only about 40% of cotton's economic production, while cottonseed accounts for about 60%. The oil content in cottonseed is about 25-39%, and it is the fifth largest edible oil source in my country, second only to soybeans, peanuts, rapeseed and sunflower. Cottonseed oil contains high unsaturated fatty acids and is easily absorbed by the human body. It is an ideal raw material for salad oil, shortening, margarine and special oils. [0003] The composition and content of fatty acids in vegetable oil determine the nutritional value and oil stability of edible oil. The fatty acids in cottonseed oil are mainly composed of palmitic acid, oleic acid and linoleic acid, among which the ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/13C12Q2600/156
Inventor 柳展基王立国陈义珍傅明川李浩刘任重
Owner VEGETABLE RES INST OF SHANDONG ACADEMY OF AGRI SCI
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