CN111961747A - Method for assisting in identifying sugar content of watermelon fruit, SNP marker and KASP marker - Google Patents
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Abstract
The invention discloses a method for assisting in identifying the sugar content of watermelon fruits, an SNP marker and a KASP marker. The invention discloses a method for identifying or assisting in identifying the sugar content of watermelon fruits, which comprises the following steps: detecting the genotype of the watermelon to be detected based on the specific SNP; the sugar content of the fruit of the CC genotype watermelon is higher than that of the AA genotype watermelon. The specific SNP is as follows (a) or (b): (a) 18785631 th nucleotide on chromosome 2 of watermelon genome; (b) the 52 th nucleotide of the DNA molecule shown in the sequence 1 of the sequence table in the watermelon genome. The method can realize the initial screening of the watermelon varieties, achieves the aim of detecting the sugar content of the watermelon fruits at the seedling stage, greatly shortens the breeding period, and has important theoretical and practical significance.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a method for assisting in identifying sugar content of watermelon fruits, an SNP marker and a KASP marker.
Background
Watermelon (Citrullus lanatus) belongs to important cucurbitaceae crops, and the yield of Chinese watermelons accounts for about 60% of the annual global yield (http:// faostat. fao. org). Watermelon is an important horticultural crop with international competitiveness and great economic growth space in china. The watermelon is originated from Africa, and the sugar content of the wild watermelon fruit of Africa is low and is only 1-3 degrees. The sugar content of modern watermelon fruits can reach 10-14 degrees. The high-sugar watermelon is the main character of human breeding.
In the watermelon germplasm resources stored in the watermelon germplasm resource library (http:// www.ars-grin. gov /), genes with excellent traits such as disease resistance and the like mostly exist in wild and semi-wild low-sugar materials. Since fruit sugar is composed of raffinose in leaves, it is stored in the fruit as sucrose through a complex network of transport and metabolism. Due to linkage drag of unfavorable characters, under the condition of no molecular marker-assisted breeding sugar content, breeding breeders dare not to easily use wild and semi-wild disease-resistant excellent resources to improve the disease resistance of parents. The early-stage screening of the variety is carried out by identifying the molecular marker closely linked with the sugar content of the watermelon fruit, the aim of molecular assisted breeding is achieved, the breeding period can be greatly shortened, the breeding efficiency is improved, and the aim of applying low-sugar wild excellent resources to the molecular improvement of main cultivars is fulfilled.
Disclosure of Invention
The invention aims to provide a method for assisting in identifying the sugar content of watermelon fruits, an SNP marker and a KASP marker.
The invention provides an application of a substance for detecting specific SNP in identification or auxiliary identification of the sugar content of watermelon fruits.
The invention also protects the application of the substance for detecting the specific SNP in watermelon breeding.
The invention also provides a method for identifying or assisting in identifying the sugar content of the watermelon fruit, which comprises the following steps: detecting the genotype of the watermelon to be detected based on the specific SNP; the sugar content of the fruit of the CC genotype watermelon is higher than that of the AA genotype watermelon.
The invention also provides a method for identifying or assisting in identifying the sugar content of the watermelon fruit, which comprises the following steps: detecting the genotype of the watermelon to be detected based on the specific SNP; the CC genotype watermelon is or is candidate for a watermelon with a high sugar phenotype, and the AA genotype watermelon is or is candidate for a watermelon with a low sugar phenotype.
The invention also provides a watermelon breeding method, which comprises the following steps: detecting the genotype of the watermelon to be detected based on the specific SNP; and (3) selecting the CC genotype watermelon for breeding.
The invention also protects a specific DNA molecule which is shown as a sequence 1 in a sequence table.
The invention also protects the application of the specific DNA molecule in identification or auxiliary identification of the sugar content of the watermelon fruit.
The invention also protects a specific primer group which consists of a primer FAM-TST2-A1, a primer HEX-TST2-A2 and a primer TST 2-C.
The invention also protects the application of the specific primer group, which is (c) or (d) as follows:
(c) identifying or assisting in identifying the sugar content of the watermelon fruits;
(d) and (5) breeding the watermelon.
Any one of the above substances for detecting a specific SNP is specifically the specific primer pair.
The method for detecting the genotype of the watermelon to be detected based on the specific SNP specifically comprises the following steps: and (3) amplifying a target fragment with the specific SNP by using the genome DNA of the watermelon to be detected as a template, and then sequencing.
The method for detecting the genotype of the watermelon to be detected based on the specific SNP specifically comprises the following steps: using the genomic DNA of the watermelon to be tested as a template, and carrying out KASP-based genotyping.
The method for detecting the genotype of the watermelon to be detected based on the specific SNP specifically comprises the following steps: and (3) carrying out KASP by using the specific primer group by using the genome DNA of the watermelon to be detected as a template.
The method for detecting the genotype of the watermelon to be detected based on the specific SNP specifically comprises the following steps:
(1) using the genome DNA of the watermelon to be detected as a template, and adopting the specific primer group to carry out KASP;
(2) and (3) after the step (1) is finished, performing fluorescence scanning to determine the genotype of the watermelon to be detected.
In the reaction system of KASP, the concentrations of FAM-TST2-A1, HEX-TST2-A2 and TST2-C were all 0.5 mM.
Reaction procedure of KASP: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 deg.C for 20s, 61-55 deg.C (touch down program is selected, and each cycle is reduced by 0.6 deg.C) for 1min, 10 cycles; denaturation at 94 ℃ for 20s, renaturation & elongation at 55 ℃ for 1min, 26 cycles.
FAM, blue (focused on the X-axis), represents the CC genotype.
HEX, red (focusing on the Y axis), represents AA genotype.
The primer FAM-TST2-A1 has the 22 nd to 49 th nucleotides in the sequence 2 of the sequence table;
the primer HEX-TST2-A2 has the 22 nd to 51 th nucleotides in the sequence 3 of the sequence table;
the primer TST2-C is shown as a sequence 4 in the sequence table.
The primer FAM-TST2-A1 is a single-stranded DNA molecule shown in a sequence 2 in a sequence table.
The primer HEX-TST2-A2 is a single-stranded DNA molecule shown in sequence 3 of the sequence table.
The primer TST2-C is specifically a single-stranded DNA molecule shown in a sequence 4 in a sequence table.
The breeding target of the watermelon breeding is to cultivate the watermelon with high sugar phenotype.
The breeding target of any one of the watermelon breeding is to improve the sugar content of watermelon fruits.
The sugar content of the fruit of the CC genotype watermelon is higher than that of the AA genotype watermelon.
The CC genotype watermelon is or is candidate for a watermelon with a high sugar phenotype, and the AA genotype watermelon is or is candidate for a watermelon with a low sugar phenotype.
Watermelon with high sugar phenotype: the fruit contains sugar of 5Brix or more.
Watermelon with a low sugar phenotype: the fruit contains less than 5Brix of sugar.
Any one of the specific SNPs is as follows (a) or (b):
(a) 18785631 th nucleotide on chromosome 2 of watermelon genome;
(b) the 52 th nucleotide of the DNA molecule shown in the sequence 1 of the sequence table in the watermelon genome;
the specific SNP is A/C polymorphism.
In scientific research and practice in the field of watermelon breeding, a Single Nucleotide Polymorphism (SNP) marker linked with the sugar content of watermelon fruits and a method for identifying the marker by using competitive Allele Specific PCR (KASP) are required.
The invention develops the SNP marker related to the sugar content of watermelon fruits, identifies the sugar content of the fruits of 111 natural watermelon population materials, utilizes the KASP marker to detect the linkage condition of the SNP marker and the sugar content of the watermelon fruits, and finds that the initial screening of watermelon varieties can be carried out through SNP polymorphism or genotype. The method can realize the initial screening of the watermelon varieties, achieves the aim of detecting the sugar content of the watermelon fruits at the seedling stage, greatly shortens the breeding period, and has important theoretical and practical significance.
Drawings
FIG. 1 is a graph showing the results of example 2.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Unless otherwise stated, the quantitative tests in the following examples were performed in triplicate, and the results were averaged.
The germplasm resources of 111 watermelons used in the examples are shown in table 1 and can be obtained from a germplasm resource library of vegetable research center of agriculture and forestry academy of sciences of Beijing. The germplasm resource numbers in the subsequent tables are all in one-to-one correspondence with the germplasm resource numbers in the table 1.
TABLE 1
Example 1 discovery of SNP marker and KASP marker relating to sugar content in watermelon fruit
Test materials: 111 kinds of watermelon germplasm resources.
First, detection of sugar content in fruit
All materials tested were tested in parallel: the sugar content of the mature fruit was measured using a hand-held refractometer (ATAGO co., ltd., Tokyo, Japan) grown continuously at the same site for two years. The average value of the fruits of the plants in two years is taken as the sugar content of the fruits of each test material. The fruit contains sugar in an amount of 5Brix or more, and is defined as a high-sugar phenotype. The fruit has a sugar content of less than 5Brix and is defined as a low sugar phenotype.
The results of the sugar content in the fruit are shown in column 2 of Table 2. 78 parts of the test material had a high sugar phenotype and 33 parts of the test material had a low sugar phenotype.
TABLE 2
Second, discovery of SNP associated with sugar content in fruit
1. Whole genome association analysis
The TASSEL3.0 software is used for carrying out whole genome association analysis on the test material, and as a result, the fruit sugar content gene is positioned at the CHR2-18785631 position and SNP on both sides of the CHR2-18785631 position.
2. Discovery of SNPs related to sugar content in fruit
Comparing genome sequences of the watermelon natural population materials in a primary positioning gene interval by using a test material whole-gene association analysis result to obtain candidate SNP sites highly conforming to the phenotypic characters of the watermelon natural population materials; searching SNP loci closely linked with the sugar content of the fruits in the natural population material of the watermelon, and finding that one SNP locus (A → C) of the No. 2 chromosome is most related to the sugar content character of the fruits.
The SNP found above was named as a specific SNP located in the watermelon genome (http:// cucurbitgenomics.org/V2) nucleus 18785631 of chromosome 2The nucleotide (corresponding to the 52 th nucleotide in the sequence 1 of the sequence table, M represents A or C) is in an A/C polymorphism.
Based on the genotype of the specific SNP, the genotype of the test material is shown in column 3 of Table 2 as AA genotype or CC genotype. The sugar content of the fruit of the CC genotype watermelon is higher than that of the AA genotype watermelon.
Design of three, KASP labeled primer group
And designing a complete set of KASP primer sets according to the specific SNP. The KASP primer group consists of 2 upstream primers (FAM-TST2-A1 and HEX-TST2-A2) and 1 universal downstream primer (TST 2-C).
FAM-TST2-A1 (SEQ ID NO: 2 of the sequence Listing):
5′-GAAGGTGACCAAGTTCATGCTCTCAAAACCCTCTTTAAATTTTACTGAC-3′；
HEX-TST2-A2 (SEQ ID NO: 3 of the sequence listing):
5′-GAAGGTCGGAGTCAACGGATTAACTCAAAACCCTCTTTAAATTTTACTGAA-3′；
TST2-C (SEQ ID NO: 4 of the sequence Listing): 5'-GAATTATCACACTGACCATTGTTTCACTC-3' are provided.
Example 2 detection of genotype and identification of sugar content in fruits Using KASP primer set
Test materials: 111 kinds of watermelon germplasm resources.
1. Taking the plant leaves of the tested material, and extracting the genome DNA.
2. Genotyping was performed by using the genomic DNA obtained in step 1 as a template and a KASP primer set by KASP (Kompetitive Allle-Specific PCR, competitive Allele-Specific PCR).
The SNP genotyping procedure was carried out according to the experimental procedure of KASP technique provided by LGC company, and the reagents, consumables and instruments used below, which are not specifically described, were all provided by LGC company, including reagent amounts, usage, and the whole experimental procedure were carried out according to the LGC company's instruction KASP user guide and manual (www.lgcgenomics.com).
Reaction system (total volume 2 μ l): template DNA (DNA content 50-100ng), 2 XKASP reaction mix 1. mu.l, mixed primer 0.2. mu.l, and the balance ddH2And O. Effective components provided by mixed primersIs divided into FAM-TST2-A1, HEX-TST2-A2 and TST 2-C. In the reaction system, the concentrations of FAM-TST2-A1, HEX-TST2-A2 and TST2-C were all 0.5 mM.
2 XKASP reaction Mix, also known as KASP 2 XMaster Mix, is a product of LGC company under the trade name KBS-1016-002. The 2 xKASP reaction mix comprises a fluorescent probe A, a fluorescent probe B, a quenching probe A and a quenching probe B, as well as high-fidelity Taq enzyme, dNTP and the like. The sequence of the fluorescent probe A is 5'-GAAGGTGACCAAGTTCATGCT-3', and the 5 ' end is connected with 1 fluorophore FAM; the sequence of the fluorescent probe B is 5'-GAAGGTCGGAGTCAACGGATT-3', and the 5 ' end is connected with 1 fluorophore HEX; the sequence of the quenching probe A is 5'-AGCATGAACTTGGTCACCTTC-3', and the 3 ' terminal is connected with a quenching group BHQ; the sequence of the quenching probe B is 5'-AATCCGTTGACTCCGACCTTC-3', and the 3 ' terminal is connected with a quenching group BHQ.
Reaction procedure: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 deg.C for 20s, 61-55 deg.C (touch down program is selected, and each cycle is reduced by 0.6 deg.C) for 1min, 10 cycles; denaturation at 94 ℃ for 20s, renaturation & elongation at 55 ℃ for 1min, 26 cycles.
3. After the step 2 is completed, when the temperature of the PCR amplification product is reduced to below 40 ℃, the fluorescence value is read through FAM and HEX light beam scanning of a microplate reader (reading value is observed when FAM fluorescence is at the wavelength of 485nm of exciting light and 520nm of emitting light; reading value is observed when HEX fluorescence is at the wavelength of 528nm of exciting light and 560nm of emitting light), and the genotype is judged according to the fluorescence signal color.
The results are shown in FIG. 1. The results are presented as scatter plots, each point representing a sample, with the same genotype points concentrated in one region. The red fluorescence signal (concentrated near the Y-axis) represents the AA genotype. The blue fluorescence signal (concentrated around the X-axis) represents the CC genotype. The green fluorescent signal represents the AC genotype.
The results of the detection were 100% consistent with the genotype results in Table 2. Watermelon of CC genotype all have high sugar phenotype. The AA genotype watermelons all have a low sugar phenotype.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Sequence listing
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Jingyan Yinong (Beijing) seed industry science and technology Co., Ltd
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Claims (10)
1. The application of the substance for detecting the specific SNP in identifying or assisting in identifying the sugar content of the watermelon fruit;
the specific SNP is as follows (a) or (b):
(a) 18785631 th nucleotide on chromosome 2 of watermelon genome;
(b) the 52 th nucleotide of the DNA molecule shown in the sequence 1 of the sequence table in the watermelon genome;
the specific SNP is A/C polymorphism.
2. Detecting the application of the substance of the specific SNP in the breeding of the watermelon;
the specific SNP is as follows (a) or (b):
(a) 18785631 th nucleotide on chromosome 2 of watermelon genome;
(b) the 52 th nucleotide of the DNA molecule shown in the sequence 1 of the sequence table in the watermelon genome;
the specific SNP is A/C polymorphism.
3. A method for identifying or assisting in identifying the sugar content of watermelon fruits comprises the following steps: detecting the genotype of the watermelon to be detected based on the specific SNP; the sugar content of the fruit of the CC genotype watermelon is higher than that of the AA genotype watermelon;
the specific SNP is as follows (a) or (b):
(a) 18785631 th nucleotide on chromosome 2 of watermelon genome;
(b) the 52 th nucleotide of the DNA molecule shown in the sequence 1 of the sequence table in the watermelon genome;
the specific SNP is A/C polymorphism.
4. A method for identifying or assisting in identifying the sugar content of watermelon fruits comprises the following steps: detecting the genotype of the watermelon to be detected based on the specific SNP; the CC genotype watermelon is or is candidate for a watermelon with a high sugar phenotype, and the AA genotype watermelon is or is candidate for a watermelon with a low sugar phenotype.
The specific SNP is as follows (a) or (b):
(a) 18785631 th nucleotide on chromosome 2 of watermelon genome;
(b) the 52 th nucleotide of the DNA molecule shown in the sequence 1 of the sequence table in the watermelon genome;
the specific SNP is A/C polymorphism.
5. A method for breeding watermelon comprises the following steps: detecting the genotype of the watermelon to be detected based on the specific SNP; selecting CC genotype watermelon for breeding;
the specific SNP is as follows (a) or (b):
(a) 18785631 th nucleotide on chromosome 2 of watermelon genome;
(b) the 52 th nucleotide of the DNA molecule shown in the sequence 1 of the sequence table in the watermelon genome;
the specific SNP is A/C polymorphism.
6. The specific DNA molecule is shown as a sequence 1 in a sequence table.
7. The use of the specific DNA molecule of claim 6 for identifying or assisting in identifying the sugar content of watermelon fruit.
8. The specific primer pair consists of a primer FAM-TST2-A1, a primer HEX-TST2-A2 and a primer TST 2-C;
the primer FAM-TST2-A1 has the 22 nd to 49 th nucleotides in the sequence 2 of the sequence table;
the primer HEX-TST2-A2 has the 22 nd to 51 th nucleotides in the sequence 3 of the sequence table;
the primer TST2-C is shown as a sequence 4 in the sequence table.
9. The specific primer pair of claim 8, wherein:
the primer FAM-TST2-A1 is a single-stranded DNA molecule shown in a sequence 2 of a sequence table.
The primer HEX-TST2-A2 is a single-stranded DNA molecule shown in a sequence 3 of a sequence table.
The primer TST2-C is a single-stranded DNA molecule shown in a sequence 4 of the sequence table.
10. The use of the specific primer pair according to claim 8 or 9, which is (c) or (d) below:
(c) identifying or assisting in identifying the sugar content of the watermelon fruits;
(d) and (5) breeding the watermelon.
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| CN113969323A (en) * | 2021-10-14 | 2022-01-25 | 广东省农业科学院蔬菜研究所 | SNP molecular marker related to luffa melon length and application thereof |
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