CN113755633A - KASP molecular marker for high-throughput detection of cotton GhFAD2-1 gene high-oleic acid mutation site - Google Patents

Abstract

The invention discloses a KASP molecular marker for high-throughput detection of cotton GhFAD2-1 gene high-oleic acid mutation sites, wherein the molecular marker linked with GhFAD2-1A nucleotide mutation sites is KASP-A429, and the molecular marker linked with GhFAD2-1D nucleotide mutation sites is KASP-D429. The method can make the large-population screening in the cotton high-oleic-acid breeding process more efficient, economic, simple and accurate.

Description

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

Technical Field

The invention relates to a KASP molecular marker for high-throughput detection of cotton GhFAD2-1 gene high-oleic acid mutation sites, and belongs to the technical field of biology.

Background

Cotton is an important fiber and oil crop. Fiber accounts for only about 40% of the economic yield of cotton, while cotton seed accounts for about 60%. The oil content in the cottonseed is about 25-39%, which is the fifth edible oil source in China and is only second to soybean, peanut, rape and sunflower. The cottonseed oil contains higher unsaturated fatty acid, is easy to be absorbed by human body, and is an ideal raw material for salad oil, shortening, margarine and special grease.

The composition and content of fatty acids in vegetable oils determine the nutritional value and oil stability of edible oils. The fatty acids in cottonseed oil consist primarily of palmitic acid, oleic acid and linoleic acid, with linoleic acid being present at a higher level of about 46.7-58.2% and oleic acid being present at a lower level of about 14.7-21.7%. Linoleic acid (C18:2) is a polyunsaturated fatty acid that is unstable, has poor thermal stability and oxidation resistance, is not shelf stable, and is susceptible to deterioration when cooked at high temperatures; oleic acid (C18:1) as monounsaturated fatty acid has high stability, is not easy to generate trans-fatty acid in the cooking process, is favored by consumers and is praised as 'safe fatty acid' by the nutritional science community. The high oleic acid (more than 70 percent) edible oil and the products thereof become the hot points of attention by virtue of the characteristics of oxidation resistance, thrombus resistance, hypertension resistance and blood fat concentration reduction. Therefore, increasing the oleic acid content of cottonseed is an important goal in improving the quality of cottonseed oil.

FAD2 gene is a key gene for controlling the content of oleic acid and linoleic acid in plant seeds, and the delta coded by the gene^12-Fatty acid desaturase catalyzes further desaturation of oleic acid (C18:1) to linoleic acid (C18: 2). Upland cotton is an allotetraploid (2 n-4X-52, genome AADD), and the genome contains 8 FAD2 homologous genes, wherein GhFAD2-1A/D is mainly expressed in seeds and is a key gene for catalyzing oleic acid to form linoleic acid (Xuxiaolan et al, 2019; Chen et al, 2021). The GhFAD2-1A and the GhFAD2-1D are homologous genes positioned on different subgenomes, both the genes contribute to the content of oleic acid and linoleic acid, and the transcription of the two genes is simultaneously inhibited or the enzyme activity is activatedThe cottonseed will show high oleic acid character. At present, 3 parts of the germplasm material with the FAD2-1D single gene natural mutation is only found in sea island cotton, and the oleic acid content is increased to 42% (Shockey et al, 2017). No naturally mutated or artificially mutagenized high oleic acid mutants have been found in upland cotton. In 2002, Liu et al utilize RNAi technology to inhibit the expression of cotton FAD2 gene, and create a new transgenic cotton material with oleic acid content as high as 77%.

In 2021, the laboratory uses cotton GhFAD2-1A/D gene as a target, 1 target spot is designed in the Fatty acid desaturase domain, pRGEB32-GhU6.9-NPT II vector is used for constructing a CRISPR/Cas9 gene editing vector of the GhFAD2-1A/D gene, cotton strain Jin668 is transformed by agrobacterium, 4 non-transgenic (without vector selection marker gene) high oleic acid materials (m1-2, m1-3, m20-2 and m27-3) are detected in the T1 generation, the oleic acid content of cotton seeds is increased from 14% to 78%, and the linoleic acid content is reduced from 59% to 7% (Chen et al, 2021). Through detection, 1T base is inserted into 429nt of a coding region of a GhFAD2-1A gene (Ghir _ A13G022660.1, HAU assembly) of m1-2, so that a frameshift mutation is generated; the coding region 429nt of the GhFAD2-1D gene (Ghir _ D13G023320.1, HAU assembly) is inserted with 1A base, and a frame shift mutation is also generated. The method takes m1-2 as a male parent, respectively hybridizes with a plurality of high-quality, high-yield and disease-resistant cotton varieties, and expects to transfer the high-oleic acid character into the current main-planting cotton variety. However, the Sanger sequencing method is used for detecting the filial generation, the detection efficiency is low, the detection period is long, the detection cost is high, and a high-throughput detection method is urgently needed in the high-oleic-acid breeding and production of cotton.

Disclosure of Invention

The invention provides molecular markers KASP-A429 and KASP-D429 for high-oleic acid mutation sites of GhFAD2-1A and GhFAD2-1D genes of cotton in a high-throughput and rapid manner aiming at a novel high-oleic acid cotton germplasm m1-2 edited and created by CRISPR/Cas9 gene. The invention designs specific amplification primers and universal primers of a GhFAD2-1A gene and a GhFAD2-1D gene, and establishes a method for detecting allelic variation of a cotton GhFAD2-1A gene and a GhFAD2-1D gene by applying an LGC high-throughput marker detection platform.

The technical scheme of the invention is as follows: a KASP molecular marker for high-throughput detection of cotton GhFAD2-1 gene high-oleic acid mutation sites is characterized in that,

the KASP molecular marker linked with the GhFAD2-1A nucleotide mutation site is KASP-A429, the nucleotide sequence is as follows: 5 '-GAAAATCAGTCACCGCCGTCA [ T/- ] CCACTCGAACACCGGTTCCATGGAGCGTGACGAAGTATTCGTGCCCAAACCCAAGTCTAAATTATCATGCTTTGCGAAATACTTAAACAATCCACCCGGTCGAGT-3' (SEQ ID No. 1);

the KASP molecular marker linked with the GhFAD2-1D nucleotide mutation site is KASP-D429, the nucleotide sequence of which is as follows: 5 '-GAAAATCAGTCACCGCCGTCA [ A/- ] CCACTCGAACACCGGTTCCATGGAGCGTGACGAAGTATTCGTGCCCAAACCCAAGTCTAAATTATCATGCTTTGCGAAATACTTCAACAATCCACCCGGTCGAG-3' (SEQ ID No. 2).

The invention also provides a KASP molecular marker primer for detecting the high oleic acid mutation site of the cotton GhFAD2-1 gene in high throughput, which is characterized in that,

1) the primer set of KASP-A429 is as follows:

the specific upstream primer GhFAD2-1A-HEX of KASP-A429 is 5'-GAAAATCAGTCACCGCCGTCAt-3' (SEQ ID No. 3);

a specific downstream primer GhFAD2-1A-R: 5'-ACTCGACCGGGTGGATTGTTT-3' (SEQ ID No.4) of KASP-A429;

a universal upstream primer: GhFAD2-1AD-FAM 5'-GAAAATCAGTCACCGCCGTCAC-3', (SEQ ID No. 5);

2) the primer set of KASP-D429 was:

a specific upstream primer GhFAD2-1D-HEX of KASP-D429: 5'-GAAAATCAGTCACCGCCGTCAa-3', (SEQ ID No. 6);

specific downstream primer GhFAD2-1D-R: 5'-CTCGACCGGGTGGATTGTTG-3' of KASP-D429 (SEQ ID No. 7);

a universal upstream primer: GhFAD2-1AD-FAM: 5'-GAAAATCAGTCACCGCCGTCAC-3', (SEQ ID No. 5).

The invention also discloses a detection kit for detecting the high oleic acid mutation sites of the GhFAD2-1 gene of cotton in a high-throughput manner (used for determining nucleotide mutation sites of 429nt of the GhFAD2-1A and GhFAD2-1D coding regions in a cotton sample), which is characterized by at least comprising PCR amplification primers shown in SEQ ID Nos. 3-7. Furthermore, the detection kit also comprises 2 XKASP Mix and cotton sample genome DNA.

The invention also discloses a method for identifying the high oleic acid character phenotype of the GhFAD2-1 gene of cotton, which is characterized by comprising the following steps:

carrying out PCR amplification on the genome DNA of the cotton sample to be detected by adopting a PCR amplification primer shown in SEQ ID No. 3-7;

the result of identifying the target InDel genotype by PCR amplification by adopting a molecular marker KASP-A429 primer group is judged as follows: if the fluorescence signal data of the amplification product of the cotton sample to be detected is close to a vertical axis (HEX signal) through analysis of Douglas genotyping software, only T is detected at the 429 th base of the GhFAD2-1A gene coding region of the cotton sample to be detected, the gene is pure and high oleic acid mutant gene, the genotype is defined as AA, if the fluorescence signal data of the amplification product of the cotton sample to be detected is close to a horizontal axis (FAM signal), the base insertion mutation is not generated at the 429 th base of the GhFAD2-1A gene coding region of the cotton sample to be detected, the gene is pure and normal oleic acid cotton (wild type), the genotype is defined as AA, if the fluorescence signal data of the amplification product of the cotton sample to be detected is located between the horizontal axis and the vertical axis (FAM signal and the HEX signal), the cotton sample is judged to be a heterozygous, and the genotype is defined as AA;

the result of identifying the target InDel genotype by PCR amplification by adopting a molecular marker KASP-D429 primer group is judged as follows: if the fluorescence signal data of the amplification product of the cotton sample to be detected is close to a vertical axis (HEX signal) through analysis of Douglas genotyping software, only A is detected at the 429 th base of the GhFAD2-1D gene coding region of the cotton sample to be detected, the gene is pure and high oleic acid mutant gene, the genotype is defined as BB, if the fluorescence signal data of the amplification product of the cotton sample to be detected is close to a horizontal axis (FAM signal), the base insertion mutation is not generated at the 429 th base of the GhFAD2-1D gene coding region of the cotton sample to be detected, the gene is pure and normal oleic acid cotton (wild type), the genotype is defined as BB, if the fluorescence signal data of the amplification product of the cotton sample to be detected is located between the horizontal axis and the vertical axis (FAM signal and the HEX signal), the cotton sample is judged to be a hybrid, and the genotype is defined as Bb;

and combining the detection results of KASP-A429 and KASP-D429, and if the GhFAD2-1A and GhFAD2-1D in the cotton sample are detected to be pure and mutated at the same time, and the genotype is AABB, the cotton sample is a high-oleic acid cotton sample.

The invention has the beneficial effects that: the detection method adopted at present is a Sanger sequencing method, the detection period is long, the cost is high, and the flux is low, but the KASP high-flux detection system of GhFAD2-1A/D InDel genotyping established by the invention can enable large-population screening in the cotton high-oleic-acid breeding process to become more efficient, economic, simple and accurate.

Drawings

FIG. 1 is a graph of the typing of 96 test cotton materials using KASP markers.

Detailed Description

The effects of the embodiments will be described below with reference to the accompanying drawings.

Example 1: development of KASP markers closely linked with high-oleic-acid trait genes GhFAD2-1A and GhFAD2-1D of cotton

Design of KASP primer

100bp flanking sequences around 429nt site of coding region of GhFAD2-1A/D gene are extracted, 3 sets of KASP primers are designed by using Primer5.0 software, and after the primers are detected by using ArrayTape platform of Douglas Scientific company, the amplification effect and polymorphism of the labeled KASP-A429 and KASP-D429 are optimal, so that the InDel difference of GhFAD2-1A and GhFAD2-1D in wild type and high oleic acid mutant types can be obviously distinguished.

The primers of the molecular marker KASP-A429 are as follows:

1) two specific primers:

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

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

2) one universal primer:

GhFAD2-1AD-FAM 5'-GAAAATCAGTCACCGCCGTCAC-3', shown as SEQ ID No. 5;

the primers of the molecular marker KASP-D429 are as follows:

1) two specific primers:

GhFAD2-1D-HEX: 5'-GAAAATCAGTCACCGCCGTCAa-3', shown as SEQ ID No. 6;

GhFAD2-1D-R: 5'-CTCGACCGGGTGGATTGTTG-3', shown as SEQ ID No. 7;

2) one universal primer:

GhFAD2-1AD-FAM: 5'-GAAAATCAGTCACCGCCGTCAC-3', shown as SEQ ID No. 5.

The KASP markers both comprise two forward primers and one reverse primer, the forward primer of KASP-A429 is GhFAD2-1AD-FAM and GhFAD2-1A-HEX, and the reverse primer is GhFAD 2-1A-R. The forward primers of KASP-D429 are GhFAD2-1AD-FAM and GhFAD2-1D-HEX, and the reverse primer is GhFAD 2-1D-R. The 3 'ends of the two forward primers are allelic variant bases, and the 5' ends are respectively connected with specific FAM and HEX fluorescent linker sequences in KASP reagent of LGC company in England.

DNA extraction

Extracting genome DNA from young and tender cotton leaves by a conventional CTAB method.

KASP reaction test

The fluorescent quantitative PCR amplification system is as follows: 2.5. mu.L of cotton sample genomic DNA (40 ng/. mu.L), 2.5. mu.L of LGC 2 XKASP Mix (Low Rox), 0.07. mu.L of primer mixture (molar concentration ratio of forward primer FAM: forward primer HEX: reverse primer R: 2: 5);

the PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 10min for 1 cycle; denaturation at 95 ℃ for 20s, annealing at 55-62 ℃ (preferably 55 ℃) for 60s, 40 cycles.

Wherein the 2 XKASP Mix consists of a fluorescent probe A, a fluorescent probe B, a quenching probe A, a quenching probe B, high fidelity Taq enzyme, dNTP and Mg²⁺And the like; the nucleotide sequence of the fluorescent probe A is as follows: 5'-GAAGGTCGGAGTCAACGGATT-3', connecting a HEX fluorescent group at the 5 ' end; the nucleotide sequence of the fluorescent probe B is as follows: 5'-GAAGGTGACCAAGTTCATGCT-3', wherein the 5 ' end is connected with a FAM fluorophore; the nucleotide sequence of the quenching probe A is as follows: 5'-AATCCGTTGACTCCGACCTTC-3', wherein the 3 ' terminal is connected with a quenching group BHQ; the nucleotide sequence of the quenching probe B is as follows: 5'-AGCATGAACTTGGTCACCTTC-3', wherein the 3 ' end is connected with a quenching group BHQ.

After the PCR is finished, the fluorescence data is read by FAM and HEX light beam scanning of an enzyme labeling instrument and then analyzed by Douglas genotyping software.

Genotyping of target InDel using molecular marker KASP-A429 primer set (FIG. 1): if the fluorescence signal data of the amplification product of the cotton sample to be detected is close to a vertical axis (HEX signal) through analysis of Douglas genotyping software, only T is detected at the 429 th base of the coding region of the GhFAD2-1A gene of the cotton sample to be detected, the gene is pure and high oleic acid mutant gene, the genotype is defined as AA, if the fluorescence signal data of the amplification product of the cotton sample to be detected is close to a horizontal axis (FAM signal), the base insertion mutation is not generated at the 429 th base of the coding region of the GhFAD2-1A gene of the cotton sample to be detected, the gene is pure and normal oleic acid cotton (wild type), the genotype is defined as AA, and if the fluorescence signal data of the amplification product of the cotton sample to be detected is located between the horizontal axis and the vertical axis (FAM signal and the HEX signal), the cotton sample is judged to be a heterozygous, and the genotype is defined as AA;

genotyping of target InDel using molecular marker KASP-D429 primer set (FIG. 1): if the fluorescence signal data of the amplification product of the cotton sample to be detected is close to a vertical axis (HEX signal) through analysis of Douglas genotyping software, only A is detected at the 429 th base of the GhFAD2-1D gene coding region of the cotton sample to be detected, the gene is pure and high oleic acid mutant gene, the genotype is defined as BB, if the fluorescence signal data of the amplification product of the cotton sample to be detected is close to a horizontal axis (FAM signal), the base insertion mutation is not generated at the 429 th base of the GhFAD2-1D gene coding region of the cotton sample to be detected, the gene is pure and normal oleic acid cotton (wild type), the genotype is defined as BB, if the fluorescence signal data of the amplification product of the cotton sample to be detected is located between the horizontal axis and the vertical axis (FAM signal and HEX signal), the cotton sample is judged to be a heterozygous, and the genotype is defined as Bb;

when the results of KASP-A429 and KASP-D429 are combined, if GhFAD2-1A and GhFAD2-1D are detected to be pure and mutated at the same time in the cotton sample, and the genotype is AABB, the sample is the high-oleic acid cotton sample.

Example 2: application of KASP marker

To verify the utility of the KASP markers of the present invention, detection assays were performed using the cotton material described in table 1. Among them, H01-H92 is Lu Cotton 418 as female parent and High oleic acid cotton mutant m1-2(Chen Yizhen, Fu Mingchuan, Li Hao, et al, High-oleic acid content, nontrangenic allometric cascade cotton (Gossypium hirsutum L.) genic by knock out of GhFAD2 genes with CRISPR/Cas9 system.plant Biotechnology Journal,2021,19:424-426.) as male parent to cross-bred F2 isolate population, H93 and H94 are High oleic acid cotton mutants m1-2 used as positive control, H95 and H96 are Lu Cotton 418 used as negative control.

TABLE 1 introduction Table of phenotype and genotype information KASP of cotton segregating population

As a result, 12 individuals among 92 individuals of the F2 isolate population were detected to have genotypes consistent with those of the positive control m1-2 by KASP analysis as shown in Table 1 and FIG. 1. Meanwhile, 96 parts of the materials described in table 1 were analyzed using Sanger sequencing method, and the results showed that: the results of the molecular marker KASP-A429 and the Sanger sequencing analysis are completely consistent, the molecular marker KASP-D429 and the samples H25, H54 and H56 have no signals, and the results of the KASP detection of the other 93 materials and the Sanger sequencing analysis are consistent. From the analysis results, it can be seen that: the high-throughput detection system enables large-population screening in the high-oleic-acid cotton breeding process to be more efficient, economical, simple and accurate.

SEQUENCE LISTING

<110> Shandong province academy of agricultural sciences

<120> KASP molecular marker for high-throughput detection of cotton GhFAD2-1 gene high-oleic acid mutation site

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<170> PatentIn version 3.3

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<213> KASP molecular marker KASP-A429 linked with GhFAD2-1A nucleotide mutation site

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gaaaatcagt caccgccgtc atccactcga acaccggttc catggagcgt gacgaagtat 60

tcgtgcccaa acccaagtct aaattatcat gctttgcgaa atacttaaac aatccacccg 120

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<213> KASP molecular marker KASP-D429 linked with GhFAD2-1D nucleotide mutation site

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gaaaatcagt caccgccgtc aaccactcga acaccggttc catggagcgt gacgaagtat 60

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actcgaccgg gtggattgtt t 21

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gaaaatcagt caccgccgtc ac 22

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ctcgaccggg tggattgttg 20

Claims (6)

1. A KASP molecular marker for high-throughput detection of cotton GhFAD2-1 gene high-oleic acid mutation sites is characterized in that,

the KASP molecular marker linked with the GhFAD2-1A nucleotide mutation site is KASP-A429, the nucleotide sequence is as follows: 5 '-GAAAATCAGTCACCGCCGTCA [ T/- ] CCACTCGAACACCGGTTCCATGGAGCGTGACGAAGTATTCGTGCCCAAACCCAAGTCTAAATTATCATGCTTTGCGAAATACTTAAACAATCCACCCGGTCGAGT-3';

the KASP molecular marker linked with the GhFAD2-1D nucleotide mutation site is KASP-D429, the nucleotide sequence of which is as follows: 5 '-GAAAATCAGTCACCGCCGTCA [ A/- ] CCACTCGAACACCGGTTCCATGGAGCGTGACGAAGTATTCGTGCCCAAACCCAAGTCTAAATTATCATGCTTTGCGAAATACTTCAACAATCCACCCGGTCGAG-3'.

2. A KASP molecular marker primer for detecting high oleic acid mutation sites of GhFAD2-1 gene of cotton in high throughput is characterized in that,

1) the primer set of KASP-A429 is as follows:

a specific upstream primer GhFAD2-1A-HEX of KASP-A429, 5'-GAAAATCAGTCACCGCCGTCAt-3';

specific downstream primer GhFAD2-1A-R of KASP-A429 is 5'-ACTCGACCGGGTGGATTGTTT-3';

a universal upstream primer: GhFAD2-1AD-FAM 5'-GAAAATCAGTCACCGCCGTCAC-3';

2) the primer set of KASP-D429 was:

a specific upstream primer GhFAD2-1D-HEX of KASP-D429, 5'-GAAAATCAGTCACCGCCGTCAa-3';

specific downstream primer GhFAD2-1D-R of KASP-D429 is 5'-CTCGACCGGGTGGATTGTTG-3';

a universal upstream primer: GhFAD2-1AD-FAM: 5'-GAAAATCAGTCACCGCCGTCAC-3'.

3. A detection kit for detecting the high oleic acid mutation site of cotton GhFAD2-1 gene in high throughput, which is characterized in that the kit at least comprises the KASP molecular marker primer of claim 2.

4. The use of the molecular marker primers of claim 2 for determining nucleotide mutation sites of the coding regions 429nt of GhFAD2-1A and GhFAD2-1D in a cotton sample.

5. The application of the molecular marker primer of claim 2 in identifying the high oleic acid trait phenotype of cotton GhFAD2-1 gene.

6. A method for identifying a high oleic acid trait phenotype of a cotton GhFAD2-1 gene, which comprises the following steps:

performing PCR amplification on the genomic DNA of a cotton sample to be detected by using the molecular marker primer as claimed in claim 2;

the result of identifying the target InDel genotype by PCR amplification by adopting a molecular marker KASP-A429 primer group is judged as follows: if the fluorescence signal data of the amplification product of the cotton sample to be detected is close to the vertical axis through analysis of Douglas genotyping software, judging that only T is detected at the 429 th base of the GhFAD2-1A gene coding region of the cotton sample to be detected, and the gene is pure and high oleic acid mutant gene, and the genotype is defined as AA, if the fluorescence signal data of the amplification product of the cotton sample to be detected is close to the horizontal axis, judging that no base insertion mutation occurs at the 429 th base of the GhFAD2-1A gene coding region of the cotton sample to be detected, the gene is pure and normal oleic acid cotton, the genotype is defined as AA, if the fluorescence signal data of the amplification product of the cotton sample to be detected is located between the horizontal axis and the vertical axis, judging that the cotton sample is a hybrid, and the genotype is defined as AA;

the result of identifying the target InDel genotype by PCR amplification by adopting a molecular marker KASP-D429 primer group is judged as follows: if the fluorescence signal data of the amplification product of the cotton sample to be detected is close to the vertical axis through analysis of Douglas genotyping software, judging that only A is detected at the 429 th base of the GhFAD2-1D gene coding region of the cotton sample to be detected, the gene is pure and high oleic acid mutant gene, the genotype is defined as BB, if the fluorescence signal data of the amplification product of the cotton sample to be detected is close to the horizontal axis, judging that no base insertion mutation occurs at the 429 th base of the GhFAD2-1D gene coding region of the cotton sample to be detected, the gene is pure and normal oleic acid cotton, the genotype is defined as BB, and if the fluorescence signal data of the amplification product of the cotton sample to be detected is located between the horizontal axis and the vertical axis, the cotton sample is judged to be a heterozygous, and the genotype is defined as Bb;

and combining the detection results of KASP-A429 and KASP-D429, and if the GhFAD2-1A and GhFAD2-1D in the cotton sample are detected to be pure and mutated at the same time, and the genotype is AABB, the cotton sample is a high-oleic acid cotton sample.

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