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Molecular marker of upland cottonseed oil content gene GhKAS1 and screening method thereof

A technology of molecular markers and screening methods, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problem of lack of genetic information of cottonseed oil, etc., to achieve clear location, convenient identification, convenient and fast detection Effect

Active Publication Date: 2018-06-15
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the research on the genetic basis of cottonseed oil has received little attention, and the genetic information about cottonseed oil is lacking, so the development of molecular markers related to the oil content of upland cotton kernels is imminent

Method used

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  • Molecular marker of upland cottonseed oil content gene GhKAS1 and screening method thereof
  • Molecular marker of upland cottonseed oil content gene GhKAS1 and screening method thereof
  • Molecular marker of upland cottonseed oil content gene GhKAS1 and screening method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The discovery of GrKAS1 gene in embodiment 1 Raymond's cotton

[0022] Using the genes related to fatty acid synthesis in the Raymond cotton genome as a reference sequence, 162 pairs of Indel primers were designed for genotype analysis, and the cotton kernel oil content of 503 upland cotton varieties in two years and four environments was used in TASSEL2.1 Based on the linear model, a gene related to cottonseed oil content was detected as the predicted KAS1 gene in Raymond cotton, named GrKAS1. Specifically, 162 pairs of Indel primers were used to carry out PCR amplification using 503 upland cotton varieties as templates. The results of electrophoresis and polyacrylic gel electrophoresis were sorted out and correlated, and it was found that the gene amplified by PCR with a pair of Indel primers was related to oil content, and the marker was named GrKAS1. The pair of Indel primers corresponds to the predicted GrKAS1 gene in Raymond cotton, and the results of the associat...

Embodiment 2

[0026] Example 2 Molecular marker of cotton kernel oil content gene GhKAS1 in upland cotton

[0027] 1. Use CottongenBlast to compare the gene to the genome database of upland cotton, find out the fragments with high homology, and determine its position on the chromosome. It can be determined that GrKAS1 has 6 copies in the upland cotton genome, which is 6 candidates Gene.

[0028] (1) Use the website http: / / www.cottongen.org / to find out the copy number and physical location of GrKAS1 in the upland cotton genome.

[0029] (2) Predict the gene structure and protein structure of 6 candidate genes, the website used is http: / / meme.ebi.edu.au / meme / cgibin / meme.cgi.

[0030] 2. Design specific primers for 6 candidate genes

[0031] (1) Find the predicted sequence of the GhKAS1 gene in the A genome, D genome, sea island cotton and upland cotton TM-1 (compare and predict whether it is the KAS1 gene in NCBI), and design primers to amplify the full length.

[0032] (2) Compare the am...

Embodiment 3

[0041] Example 3 Association analysis of molecular marker mutation sites of cotton kernel oil content gene GhKAS1 in upland cotton

[0042] 1. Extreme material resequencing to determine the mutation site

[0043] From 503 upland cotton varieties, 10 cotton kernels with extreme oil content were selected to extract DNA, among which 10 cotton kernels with extreme oil content included 5 low-oil materials and 5 high-oil materials, and then 10 cotton kernels with extreme oil content were selected. The GhKAS1 gene in each extreme material was resequenced, and the resequencing data were processed with DNAMANV6 software. Compare sequence differences, identify mutation sites, and determine mutation sites;

[0044] Specifically: extract the DNA of 10 extreme material seeds, and use this as a template to amplify the GhKAS1 gene. Because the GhKAS1 gene is too long, primers were designed to amplify in segments, and then TA cloned. The system for TA cloning is 5 microliter system: 2.5 mi...

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Abstract

The invention provides a molecular marker of an upland cottonseed oil content gene GhKAS1 and a screening method thereof. The screening method comprises the steps of designing an Indel primer for carrying out genotyping on 503 parts of upland cotton natural species, and combining with two-year four-environment oil content phenotypic data to detect a gene relevant to cottonseed oil content, whereinthe gene is GrKAS1 predicted in raymond cotton; searching six copies of the GrKAS1 gene in an upland cotton genome, adopting the copies as templates for designing primers, and carrying out electrophoretic analysis and association analysis on the 503 parts of upland cotton species through a polypropylene gel; sequencing GhKAS1 genes in 10 parts of extreme materials in the upland cotton species, finding out a mutation site, analyzing the 503 parts of upland cotton resource groups through the self-designing primers, and utilizing the polypropylene gel for carrying out electrophoretic analysis and association analysis. The invention explores the molecular marker relevant to the cottonseed oil content, and provides a reference base for molecular marker-assisted selective breeding of cottonseedoil.

Description

technical field [0001] The invention relates to the technical field of cotton molecular breeding, in particular to a molecular marker of the cotton kernel oil content gene GhKAS1 of upland cotton, a screening method and application thereof. Background technique [0002] The two major products of cotton are fiber and cottonseed, among which the production of cottonseed oil is second only to soybean oil and rapeseed oil (Shang, et al., 2017). China, the United States, and Pakistan are the world's largest cotton producers. Cottonseed oil occupies an important position in the vegetable oil market in developed countries. The oil content in cottonseed accounts for a large proportion, and the oil content in upland cotton is 25%-40%. Cottonseed oil contains 70% unsaturated fatty acids, of which 50% is linoleic acid. Eating refined cottonseed oil can lower cholesterol and soften blood vessels, and regulate the body's metabolism (Alfred et al., 2012). The National Cotton Council o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/156C12Q2600/13
Inventor 林忠旭张献龙刘欣欣赵文霞
Owner HUAZHONG AGRI UNIV
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