CN110656199A - SNP marker and KASP marker related to watermelon fruit raffinose unloading capacity - Google Patents
SNP marker and KASP marker related to watermelon fruit raffinose unloading capacity Download PDFInfo
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Abstract
The invention discloses an SNP marker and a KASP marker related to the unloading capacity of raffinose in watermelon fruits. The invention firstly discloses the application of a substance for detecting the polymorphism or genotype of an SNP marker CHR4-12223999 and/or an SNP marker CHR4-12224010 in identifying or assisting in identifying the raffinose unloading capacity of watermelon fruits; the SNP markers CHR4-12223999 and CHR4-12224010 are respectively located at position 12223999 and position 12224010 on chromosome 4 of the watermelon genome, and are both A or G. The invention further discloses a method for identifying or assisting in identifying the raffinose unloading capacity of the watermelon fruit. The SNP marker related to the unloading capacity of the raffinose of the watermelon fruits can be used for carrying out initial screening on watermelon varieties, the aim of detecting the unloading capacity of the raffinose of the watermelon fruits at a seedling stage is fulfilled, the breeding period is shortened, and the method has important significance.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an SNP marker and a KASP marker related to the cotton seed sugar unloading capacity of watermelon fruits.
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 ℃; 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 cotton seed sugar unloading capability, breeding breeders dare not to easily use wild and semi-wild disease-resistant excellent resources to improve parent disease resistance. By identifying the gene of the raffinose unloading capability of the watermelon fruit and developing the closely linked molecular markers for the initial screening of varieties, 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.
Therefore, there is a need in the scientific research and practice in the field of watermelon breeding to propose a Single Nucleotide Polymorphism (SNP) marker linked to the raffinose-unloading capability gene of watermelon fruit and a method for identification using competitive Allele Specific PCR (KASP) marker.
Disclosure of Invention
The invention aims to solve the technical problem of how to identify the unloading capacity of the raffinose of the watermelon fruit so as to breed the watermelon variety with strong unloading capacity of the raffinose of the watermelon fruit.
In order to solve the technical problems, the invention firstly provides the application of a substance for detecting the polymorphism or genotype of the SNP marker CHR4-12223999 and/or the SNP marker CHR4-12224010 in identifying or assisting in identifying the raffinose unloading capacity of the watermelon fruit;
the SNP marker CHR4-12223999 is located at 12223999 th nucleotide on No. 4 chromosome of the watermelon genome, and is A or G;
the SNP marker CHR4-12224010 is located at 12224010 th nucleotide on No. 4 chromosome of the watermelon genome, and is A or G.
The application of the substance for detecting the polymorphism or genotype of the SNP marker CHR4-12223999 and/or the SNP marker CHR4-12224010 in watermelon breeding is also within the protection scope of the invention.
In the application, the watermelon breeding is to culture a watermelon variety with strong cotton seed sugar unloading capability of watermelon fruits.
In the invention, the SNP marker CHR4-12223999 corresponds to the 47 th nucleotide of a sequence 1 in a sequence table, namely r represents A or G; the SNP marker CHR4-12224010 corresponds to the 58 th nucleotide of the sequence 1 in the sequence table, namely r represents A or G.
In the application, the detection of the polymorphism or the genotype of the SNP marker CHR4-12223999 and/or the SNP marker CHR4-12224010 can be specifically used for detecting the nucleotide type of the SNP marker CHR4-12223999 and/or the SNP marker CHR4-12224010 in watermelon genome. The genotype of the SNP marker CHR4-12223999 is AA (AA genotype for short), GG (GG genotype for short) or AG (AG genotype for short); the AA genotype is the homozygous type of the SNP marker CHR4-12223999 in the watermelon genome being A, the GG genotype is the homozygous type of the SNP marker CHR4-12223999 in the watermelon genome being G, and the AG genotype is the heterozygous type of the SNP marker CHR4-12223999 in the watermelon genome being A and G. The genotype of the SNP marker CHR4-12224010 is AA (AA genotype for short), GG (GG genotype for short) or AG (AG genotype for short); the AA genotype is the homozygous type of the SNP marker CHR4-12224010 in the watermelon genome being A, the GG genotype is the homozygous type of the SNP marker CHR4-12224010 in the watermelon genome being G, and the AG genotype is the heterozygous type of the SNP marker CHR4-12224010 in the watermelon genome being A and G.
In the application, the substance for detecting the polymorphism or the genotype of the SNP marker CHR4-12223999 and/or the SNP marker CHR4-12224010 comprises a substance A for detecting the polymorphism or the genotype of the SNP marker CHR4-12223999 and/or a substance B for detecting the polymorphism or the genotype of the SNP marker CHR 4-12224010;
the substance A is a set of primers for amplifying watermelon genome DNA fragments including the SNP marker CHR 4-12223999;
the substance B is a set of primers for amplifying watermelon genome DNA fragments including the SNP marker CHR 4-12224010.
In the above application, the substance A includes substances used in A1) or A2) or A3) as follows:
A1) the primer set consists of a single-stranded DNA molecule or a derivative thereof shown in a sequence 2 in a sequence table, a single-stranded DNA molecule or a derivative thereof shown in a sequence 3 in the sequence table and a single-stranded DNA molecule shown in a sequence 4 in the sequence table;
A2) PCR reagents containing a 1);
A3) a kit comprising a1) or a 2);
the substance B comprises B1) or B2) or B3) as follows:
B1) the primer set consists of a single-stranded DNA molecule or a derivative thereof shown in a sequence 5 in a sequence table, a single-stranded DNA molecule or a derivative thereof shown in a sequence 6 in the sequence table and a single-stranded DNA molecule shown in a sequence 7 in the sequence table;
B2) PCR reagents containing B1);
B3) a kit comprising B1) or B2).
In the application, the derivative of the single-stranded DNA molecule shown in the sequence 2 in the sequence table is obtained by connecting the 5' end of the single-stranded DNA molecule shown in the sequence 2 with a fluorescent label A;
wherein, the fluorescent label A in the derivative of the single-stranded DNA molecule shown in the sequence 2 in the sequence table can be any fluorescent label in the field, for example, FAM or HEX;
in a particular embodiment of the present invention,the derivative of the single-stranded DNA molecule shown in the sequence 2 in the sequence table is HEX-AGA2-X1, and the nucleotide sequence of the derivative is 5-GAAGGTCGGAGTCAACGGATTGGTTTTCTTCTAAAGTTCAAACCCTTG-3' (underlined sequence is the fluorescent tag HEX).
The derivative of the single-stranded DNA molecule shown in the sequence 3 in the sequence table is obtained by connecting the 5' end of the single-stranded DNA molecule shown in the sequence 3 with a fluorescent label B.
Wherein, the fluorescent label in the derivative of the single-stranded DNA molecule shown in sequence 3 in the sequence table can be any fluorescent label different from fluorescent label A in the field, for example, HEX or FAM.
In the specific embodiment of the invention, the derivative of the single-stranded DNA molecule shown in the sequence 3 in the sequence table is specifically FAM-AGA2-Y1, and the nucleotide sequence thereof is 5-GAAGGTGACCAAGTTCATGCTTAGGTTTTCTTCTAAAGTTCAAACCCTTA-3' (underlined sequence is fluorescent tag FAM).
In the application, the derivative of the single-stranded DNA molecule shown in the sequence 5 in the sequence table is obtained by connecting the 5' end of the single-stranded DNA molecule shown in the sequence 5 with a fluorescent label C;
wherein, the fluorescent label C in the derivative of the single-stranded DNA molecule shown in the sequence 5 in the sequence table can be any fluorescent label in the field, for example, FAM or HEX;
in the specific embodiment of the invention, the derivative of the single-stranded DNA molecule shown in the sequence 5 in the sequence table is HEX-AGA2-X2, and the nucleotide sequence is 5-GAAGGTCGGAGTCAACGGATTGATTGTCTCTTCATTGGACATCACC-3' (underlined sequence is the fluorescent tag HEX).
The derivative of the single-stranded DNA molecule shown in the sequence 6 in the sequence table is obtained by connecting the 5' end of the single-stranded DNA molecule shown in the sequence 6 with a fluorescent label D.
Wherein, the fluorescent label D in the derivative of the single-stranded DNA molecule shown in the sequence 6 in the sequence table can be any fluorescent label different from the fluorescent label C in the field, for example, HEX or FAM.
In a specific embodiment of the present invention, the sequence listing isThe derivative of the single-stranded DNA molecule shown as the sequence 6 is specifically FAM-AGA2-Y2, and the nucleotide sequence of the derivative is 5-GAAGGTGACCAAGTTCATGCTAGATTGTCTCTTCATTGGACATCACT-3' (underlined sequence is fluorescent tag FAM). The invention further provides a method for identifying or assisting in identifying the raffinose unloading capacity of the watermelon fruits.
The method for identifying or assisting in identifying the raffinose unloading capacity of the watermelon fruits comprises the following steps of: detecting the genotype of the SNP marker CHR4-12223999 and/or the SNP marker CHR4-12224010 in the genome of the watermelon to be detected, and identifying or assisting in identifying the raffinose unloading capacity of the watermelon fruit to be detected according to the genotype;
the SNP marker CHR4-12223999 is a watermelon to be detected with the genotype of GG, the raffinose unloading capability of the watermelon fruit to be detected is stronger than or is candidate to be stronger than that of the SNP marker CHR4-12223999, and the genotype of the watermelon to be detected is AA; wherein, the AA genotype is the homozygous type of the SNP marker CHR 4-12223999A in the watermelon genome, and the GG genotype is the homozygous type of the SNP marker CHR 4-12223999G in the watermelon genome;
the SNP marker CHR4-12224010 is a watermelon to be detected with the genotype of GG, the raffinose unloading capability of the watermelon fruit to be detected is stronger than or is candidate to be stronger than that of the SNP marker CHR4-12224010, and the genotype of the watermelon to be detected is AA; wherein, the AA genotype is the homozygous type of the SNP marker CHR 4-12224010A in the watermelon genome, and the GG genotype is the homozygous type of the SNP marker CHR 4-12224010G in the watermelon genome.
The invention also provides a breeding method of the watermelon.
The breeding method of the watermelon comprises the following steps: detecting the genotype of the SNP marker CHR4-12223999 and/or the SNP marker CHR4-12224010 in the genome of the watermelon to be detected, and selecting the watermelon with the genotype of the SNP marker CHR4-12223999 in the genome of the watermelon to be detected being GG and/or the SNP marker CHR4-12224010 being GG for breeding;
the genotype of the SNP marker CHR4-12223999 in the watermelon genome to be detected is the homozygous type of the SNP marker CHR4-12223999 in the watermelon genome which is GG;
the genotype of the SNP marker CHR4-12224010 in the watermelon genome to be detected is GG which is homozygous for the SNP marker CHR4-12223999 in the watermelon genome which is G.
The invention also provides a method for detecting the genotype of the SNP marker CHR4-12223999 and/or the SNP marker CHR4-12224010 in the watermelon genome to be detected.
The method for detecting the genotype of the SNP marker CHR4-12223999 and/or the SNP marker CHR4-12224010 in the watermelon genome to be detected comprises the following steps A) or B):
A) direct sequencing;
B) and (3) carrying out PCR reaction on the watermelon genome DNA to be detected by using the substance for detecting the genotype of the SNP marker CHR4-12223999 and/or the SNP marker CHR4-12224010 to obtain a product, and carrying out genotyping on the product.
In the above-mentioned method, the first step of the method,
the direct sequencing comprises the steps of detecting the nucleotide types of the SNP marker CHR4-12223999 and/or the SNP marker CHR4-12224010 in the watermelon genome to be detected: the SNP marker CHR4-12223999 is homozygous for A, and the genotype of the SNP marker CHR4-12223999 is AA (AA genotype for short); the SNP marker CHR4-12223999 is homozygous for G, and the genotype of the SNP marker CHR4-12223999 is GG (GG genotype for short); the SNP marker CHR4-12223999 is a hybrid type of A and G, and the genotype of the SNP marker CHR4-12223999 is AG (AG genotype for short);
the SNP marker CHR4-12224010 is homozygous for A, and the genotype of the SNP marker CHR4-12224010 is AA (AA genotype for short); the SNP marker CHR4-12224010 is homozygous for G, and the genotype of the SNP marker CHR4-12224010 is GG (GG genotype for short); and if the SNP marker CHR4-12224010 is a hybrid type of A and G, the genotype of the SNP marker CHR4-12224010 is AG (simply referred to as AG genotype).
The genotyping method comprises detecting the fluorescent signal of the product, determining the genotype according to the fluorescent signal: if the product only shows the color of the fluorescent label connected to the 5' end of the DNA molecule shown in the sequence 2, the genotype of the SNP marker CHR4-12223999 in the watermelon genome to be detected is GG; if the product only shows the color of the fluorescent label connected to the 5' end of the DNA molecule shown in the sequence 3, the genotype of the SNP marker CHR4-12223999 in the watermelon genome to be detected is AA; if the product only shows the color of the DNA molecule 5 'end connected with the fluorescent label shown in the sequence 2 and the superimposed color of the DNA molecule 5' end connected with the fluorescent label shown in the sequence 3, the genotype of the SNP marker CHR4-12223999 in the watermelon genome to be detected is AG;
if the product only shows the color of the fluorescent label connected to the 5' end of the DNA molecule shown in the sequence 5, the genotype of the SNP marker CHR4-12224010 in the watermelon genome to be detected is GG; if the product only shows the color of the fluorescent label connected to the 5' end of the DNA molecule shown in the sequence 6, the genotype of the SNP marker CHR4-12224010 in the watermelon genome to be detected is AA; and if the product only shows the color of the DNA molecule 5 'end connected with the fluorescent label shown in the sequence 5 and the superimposed color of the DNA molecule 5' end connected with the fluorescent label shown in the sequence 6, the genotype of the SNP marker CHR4-12224010 in the watermelon genome to be detected is AG.
The invention also provides a product.
The product is the substance for detecting the polymorphism or the genotype of the SNP marker CHR4-12223999 and/or the SNP marker CHR 4-12224010;
it has at least one of the following functions 1) or 2) or 3):
1) identifying or assisting in identifying the raffinose unloading capacity of the watermelon fruits;
2) breeding the watermelon with strong raffinose unloading capacity;
3) and (4) breeding the watermelon with strong raffinose unloading capacity.
In the invention, the watermelon fruit raffinose unloading capacity refers to the capacity of watermelon fruits to decompose raffinose into sucrose and galactose; when the content of the raffinose in the watermelon fruits is more than or equal to 0.15mg/g of fresh weight, the unloading capacity of the raffinose in the watermelon fruits is weak; when the content of the raffinose in the watermelon fruits is less than 0.15mg/g of fresh weight, the unloading capacity of the raffinose in the watermelon fruits is strong.
The invention develops the SNP marker related to the unloading capacity of the raffinose of the watermelon fruits, identifies the flavor of the fruits of 115 watermelon natural population materials, and utilizes the KASP marker to detect the linkage condition of the SNP marker and the unloading capacity of the raffinose of the watermelon fruits, finds that the watermelon varieties can be initially screened through SNP polymorphism or genotype, achieves the aim of detecting the unloading capacity of the raffinose of the watermelon fruits at the seedling stage, establishes a molecular marker assisted breeding system for screening the unloading capacity of the raffinose of the watermelon fruits, greatly shortens the breeding period and has important theoretical and practical significance.
Drawings
FIG. 1 is a graph showing the results of the location of gene AGA2 for the strong and weak ability to unload raffinose from watermelon fruit in example 1.
FIG. 2 shows the results of genotyping 115 natural watermelon population materials using the set of KASP-labeled primers (HEX-AGA2-X1, FAM-AGA2-Y1 and AGA2-C1) obtained in example 2, wherein the left side of the figure is red and the right side is blue.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 discovery of SNP site and KASP marker of watermelon fruit raffinose-unloading Gene AGA2
1. Test material selection
The test materials included 115 parts of watermelon natural population material (table 1);
the test material in this example was obtained from germplasm resources library of vegetable research center of agroforestry academy of sciences of Beijing.
2. Determination of 115 parts watermelon natural population material fruit raffinose unloading capacity
Adopting ultra-high-performance liquid chromatography (UHPLC) to identify raffinose of 115 watermelon natural population materials, continuously planting each plant line for 3 years, and utilizing data of different field phenotypes in 3 years, wherein the average value is the final raffinose content of each plant line material. The 115 watermelon natural population materials shown in table 1 were subjected to raffinose content identification, and any of the following cases was defined as weak raffinose-unloading ability: the content of the raffinose in the fruits is more than or equal to 0.15mg/g fresh weight. Any of the following conditions is defined as strong raffinose unloading capacity: the content of the raffinose in the fruits is less than 0.15mg/g fresh weight. The results are shown in Table 1, 92 parts material raffinose having a strong unloading capacity and 23 parts material raffinose having a weak unloading capacity.
Table 1115 natural watermelon populations' fruit raffinose-unloading abilities and genotypes
3. Whole genome association analysis
The total genome correlation analysis of 115 watermelon natural population materials is carried out by using TASSEL3.0 software, the result is shown in figure 1, and the raffinose content gene of the fruit is positioned in CHR 4: 12223999 and SNPs flanking it, which are located in the promoter region of the AGA2 gene.
4. Acquisition of candidate SNPs
Utilizing 115 parts of watermelon natural population material whole gene correlation analysis results to carry out genome sequence comparison on the watermelon natural population material in a primary positioning gene interval so as to obtain candidate SNP sites highly conforming to the phenotypic characters of the watermelon natural population material; searching SNP sites closely linked with the raffinose content in the watermelon natural population material, and finding that two SNP sites (CHR4-12223999 and CHR 4-12224010; A → G) are linked with the raffinose content in the promoter interval of the AGA2 gene. As shown in figure 1 and table 1.
Therefore, the SNP sites related to the raffinose content of the watermelon fruits are the SNP marker CHR4-12223999 and the SNP marker CHR412224010, located in the watermelon genome respectivelyhttp://cucurbitgenomics.org/V1) at positions 12223999 and 12224010 on chromosome 4, which are A or G, corresponding to nucleotides 47 and 58, respectively, of sequence 1 in the sequence listing.
The genotype of the SNP marker CHR4-12223999 is AA (AA genotype for short), GG (GG genotype for short) or AG (AG genotype for short); the AA genotype is the homozygous type of the SNP marker CHR4-12223999 in the watermelon genome being A, the GG genotype is the homozygous type of the SNP marker CHR4-12223999 in the watermelon genome being G, and the AG genotype is the heterozygous type of the SNP marker CHR4-12223999 in the watermelon genome being A and G.
The genotype of the SNP marker CHR4-12224010 is AA (AA genotype for short), GG (GG genotype for short) or AG (AG genotype for short); the AA genotype is the homozygous type of the SNP marker CHR4-12224010 in the watermelon genome being A, the GG genotype is the homozygous type of the SNP marker CHR4-12224010 in the watermelon genome being G, and the AG genotype is the heterozygous type of the SNP marker CHR4-12224010 in the watermelon genome being A and G.
The genotype of the SNP marker CHR4-12223999 is that the raffinose unloading capacity of the watermelon variety of GG is stronger than that of the watermelon variety of AA of the SNP marker CHR 4-12223999.
The genotype of the SNP marker CHR4-12224010 is that the raffinose unloading capacity of the watermelon variety of GG is stronger than that of the watermelon variety of AA of the SNP marker CHR 4-12224010.
The invention takes the nucleotide shown in the sequence 1 where the two SNP sites (namely SNP marker CHR4-12223999 and SNP marker CHR4-12224010) are located as KASP marker. Therefore, the watermelon to be detected is determined to belong to the watermelon variety with strong or weak raffinose unloading capacity according to the two SNP loci, and the method comprises the following steps: detecting the genotype of the SNP marker CHR4-12223999 and/or the SNP marker CHR4-12224010 in the genome of the watermelon to be detected, if the genotype of the SNP marker CHR4-12223999 and/or the SNP marker CHR4-12224010 of the watermelon to be detected is GG, the watermelon to be detected is or is a variety with strong raffinose unloading capacity, and if the genotype of the SNP marker CHR4-12223999 and/or the SNP marker CHR4-12224010 of the watermelon to be detected is AA, the watermelon to be detected is or is a variety with weak raffinose unloading capacity.
Example 2 application of SNP sites of AGA2 in detection of raffinose unloading capacity of watermelon fruits
Design of first, KASP labeled primer
KASP marker primers were designed based on the SNP sites obtained in example 1:
designing a set of KASP marker primers according to the nucleotide sequence of the SNP marker CHR4-12223999, wherein the set of KASP marker primers comprises 2 upstream primers and 1 universal downstream primer, the upstream primers are DNA molecules with fluorescent labels FAM added at the 5 'ends and HEX added at the 5' ends, and the universal downstream primer is composed of single-stranded DNA molecules shown in a sequence 4 in a sequence table;
the DNA molecule with the fluorescent label HEX added at the 5 'end is obtained by adding the fluorescent label HEX at the 5' end of the single-stranded DNA molecule shown in the sequence 2, namely HEX-AGA 2-X1;
the DNA molecule with the fluorescence label FAM added at the 5 'end is obtained by adding the fluorescence label FAM at the 5' end of the single-stranded DNA molecule shown in the sequence 3, namely FAM-AGA 2-Y1;
wherein, the primer HEX-AGA2-X1 and AGA2-C1 are combined to detect GG genotype; the primers FAM-AGA2-Y1 and AGA2-C1 are combined to detect the AA genotype; the primer HEXAGA2-X1, FAM-AGA2-Y1 and AGA2-C1 are combined to detect the AG genotype.
The KASP marker primers of the SNP marker CHR4-12223999 are as follows:
AGA2-X1 (upstream primer): 5'-GGTTTTCTTCTAAAGTTCAAACCCTTG-3' (SEQ ID NO: 2);
AGA2-Y1 (upstream primer): 5'-TAGGTTTTCTTCTAAAGTTCAAACCCTTA-3' (SEQ ID NO: 3);
AGA2-C1 (downstream primer): 5 '-AATCGAGA 2 TTGTCTCTTCATTGGACATC-3' (SEQ ID NO: 4);
fluorescent label FAM 5'-GAAGGTGACCAAGTTCATGCT-3';
fluorescent label HEX is 5'-GAAGGTCGGAGTCAACGGATT-3';
HEX-AGA2-X1：
5′-GAAGGTCGGAGTCAACGGATTGGTTTTCTTCTAAAGTTCAAACCCTTG-3' (underlined sequence is fluorescent tag HEX);
FAM-AGA2-Y1：
5′-GAAGGTGAGCAAGTTCATGCTTAGGTTTTCTTCTAAAGTTCAAACCCTTA-3' (underlined sequence is fluorescent tag FAM).
Designing a set of KASP marker primers according to the nucleotide sequence of the SNP marker CHR4-12224010, wherein the set of KASP marker primers comprises 2 upstream primers and 1 universal downstream primer, the upstream primers are DNA molecules with fluorescent labels FAM added at the 5 'ends and HEX added at the 5' ends, and the universal downstream primer is composed of single-stranded DNA molecules shown as a sequence 7 in a sequence table;
the DNA molecule with the fluorescent label HEX added at the 5 'end is obtained by adding the fluorescent label HEX at the 5' end of the single-stranded DNA molecule shown as the sequence 5, namely HEXAGA 2-X2;
the DNA molecule with the fluorescence label FAM added at the 5 'end is obtained by adding the fluorescence label FAM at the 5' end of the single-stranded DNA molecule shown in the sequence 6, namely FAM-AGA 2-Y2;
wherein, the primer HEX-AGA2-X2 and AGA2-C2 are combined to detect GG genotype; the primers FAM-AGA2-Y2 and AGA2-C2 are combined to detect the AA genotype; the combination of the primers HEX-AGA2-X2, FAM-AGA2-Y2 and AGA2-C2 detects the AG genotype.
The KASP marker primers of the SNP marker CHR4-12224010 are as follows:
AGA2-X2 (upstream primer): 5'-GATTGTCTCTTCATTGGACATCACC-3' (SEQ ID NO: 5);
AGA2-Y2 (upstream primer): 5'-AGATTGTCTCTTCATTGGACATCACT-3' (SEQ ID NO: 6);
AGA2-C2 (downstream primer): 5'-GAAAATTAGGTTTTCTTCTAAAGTTCAAACCC-3' (SEQ ID NO: 7);
fluorescent label FAM 5'-GAAGGTGACCAAGTTCATGCT-3';
fluorescent label HEX is 5'-GAAGGTCGGAGTCAACGGATT-3';
HEX-AGA2-X2：
5′-GAAGGTCGGAGTCAACGGATTGATTGTCTCTTCATTGGACATCACC-3' (underlined sequence is fluorescent tag HEX);
FAM-AGA2-Y2：
5′-GAAGGTGACCAAGTTCATGCTAGATTGTCTCTTCATTGGACATCACT-3' (underlined sequence is fluorescent tag FAM).
Secondly, detecting whether the watermelon is a variety with strong raffinose unloading capability
1. Extracting genome DNA:
genomic DNA of the watermelon natural population in example 1 is extracted respectively;
the DNA extraction method is improved on the basis of the method (Murray M, Thompson W.F. Rapid isolation of high molecular weight plant DNA [ J ]. Nucl Acid Res, 1980, 8: 668-673.) referred to Murray et al (1980); the method comprises the following specific steps:
i. grinding 1.5 g of leaves of the natural population material of each watermelon into powder in liquid nitrogen, adding 9ml of 2% CTAB extracting solution (2% CTAB, 1.4mM NaCl, 100mM Tris-HCl pH8.0, 20mM EDTA pH8.0, 1% PVP-40, 0.2% beta-mercaptoethanol), mixing uniformly, and carrying out water bath at 65 ℃ for 1 hour to obtain a mixture A;
stopping the water bath of the mixture A, adding 1/3 volumes of 5M potassium acetate solution, mixing uniformly, and carrying out ice bath for 20 minutes; adding equal volume of chloroform/isoamyl alcohol (24: 1) and extracting twice to obtain supernatant A;
add 2/3 volumes of isopropanol to supernatant a to precipitate DNA; washing with washing buffer (75% ethanol, 10mM ammonium acetate) once, blow-drying, and dissolving with TE buffer (10mM Tris-HCl, 1mM EDTA, pH7.4) to obtain solution A;
iv, adding RNaseA into the solution A to enable the final concentration of the RNaseA to reach 100 mu g/ml, and uniformly mixing the RNaseA and the solution A in a water bath at 37 ℃ for 1 hour; extracting with equal volume of chloroform/isoamyl alcohol (24: 1) to obtain supernatant B;
v. adding 1/2 volumes of 7.5M ammonium acetate and 2 volumes of absolute ethyl alcohol into the supernatant B to obtain DNA precipitate;
vi, washing DNA precipitate with 70% ethanol, drying, and adding appropriate amount of ddH2Dissolving DNA to obtain respective genome DNA; the concentration of each genomic DNA was measured by OD260 using an ultraviolet spectrophotometer (Shimadzu UV-1201, Japan), and the extraction quality of each genomic DNA was checked by 1.2% agarose gel electrophoresis.
2. PCR amplification and genotype detection
1) Using the genomic DNA of each material extracted in the above step 1 as a template, PCR amplification was carried out using a set of mixed primers of KASP-labeled primers HEX-AGA2-X1, FAM-AGA2-Y1 and AGA2-C1, and genotype was examined by LGC (laboratory of the Government Chemist) experimental platform in England: if the combination of the primers HEX-AGA2-X1 and AGA2-C1 completely matches with the genomic DNA, the fluorescence signal of the detection result is dark red, which indicates that the SNP marker CHR4-12223999 is GG genotype; if the combination of the primers FAM-AGA2-Y1 and AGA2-C1 completely matches with the genomic DNA, the fluorescence signal of the detection result is blue, which indicates that the SNP marker CHR4-12223999 is of an AA genotype; if the fluorescence signal is green as a result of detection, the SNP marker CHR4-12223999 is AG genotype.
Using the genomic DNA of each material extracted in the above step 1 as a template, PCR amplification was carried out using a set of mixed primers of KASP-labeled primers HEX-AGA2-X2, FAM-AGA2-Y2 and AGA2-C2, and genotype was examined by LGC (laboratory of the Government Chemist) experimental platform in England: if the combination of the primers HEX-AGA2-X2 and AGA2-C2 completely matches with the genomic DNA, the fluorescence signal of the detection result is dark red, which indicates that the SNP marker CHR4-12224010 is GG genotype; if the combination of the primers AGA2-C2 and FAM-AGA2-Y2 completely matches with the genomic DNA, the fluorescence signal of the detection result is blue, which indicates that the SNP marker CHR4-12224010 is of an AA genotype; if the fluorescence signal is green as a result of detection, the SNP marker CHR4-12224010 is AG genotype.
2) The reaction system of the PCR amplification reaction is as follows: 0.25. mu.L of 10 XBuffer; 0.25. mu.L of dNTPs at a concentration of 2.5 mM; 0.1U Taq DNA polymerase; 0.2. mu.L of 10mM mixed primer (0.1. mu.L of each of the upstream and downstream primers); mu.L of 50 ng/. mu.L template DNA; ddH2O to 2 μ L; the final concentration of each primer in each PCR reaction system was 0.5 mM.
The reaction procedure of the PCR amplification reaction is as follows: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20s, denaturation at 61-55 ℃ (touch down program is selected, reduction of 0.6 ℃ per cycle) is carried out, 1min is carried out, and amplification is carried out for 10 cycles; denaturation at 94 ℃ for 20s, renaturation at 55 ℃ and extension for 1min, and amplification is continued for 26 cycles.
3) And (3) after the step 2) is finished, when the temperature of the PCR amplification product is reduced to be below 40 ℃, scanning and reading a fluorescence value through FAM and HEX light beams of a microplate reader (reading value of a FAM fluorescent label sequence is observed under the wavelength of 485nm of excitation light and 520nm of emission light, reading value of an HEX fluorescent label sequence is observed under the wavelength of 528nm of excitation light and 560nm of emission light), and judging the genotype of the 115 watermelon natural population materials based on the SNP markers CHR4-12223999 and/or the SNP markers CHR4-12224010 according to the color of a fluorescence signal. The specific judgment principle is as follows: if a certain watermelon natural population material shows a red fluorescent signal based on the SNP marker CHR4-12223999, the genotype of the watermelon natural population material based on the SNP marker CHR4-12223999 is GG; if a certain watermelon natural population material shows a blue fluorescent signal based on the SNP marker CHR4-12223999, the genotype of the SNP marker CHR4-12223999 is AA; if a certain watermelon natural population material shows a green signal based on the SNP marker CHR4-12223999, the genotype of the watermelon natural population material based on the SNP marker CHR4-12223999 is AG.
If a certain watermelon natural population material shows a red fluorescent signal based on the SNP marker CHR4-12224010, the genotype of the watermelon natural population material based on the SNP marker CHR4-12224010 is GG; if a certain watermelon natural population material shows a blue fluorescent signal based on the SNP marker CHR4-12224010, the genotype of the SNP marker CHR4-12224010 is AA; if a certain watermelon natural population material shows a green signal based on the SNP marker CHR4-12224010, the genotype of the watermelon natural population material based on the SNP marker CHR4-12224010 is AG.
4) Evaluation of efficiency
The results of testing 115 natural population materials (3 replicates for each watermelon material with weak unloading capacity and 1 replicate for watermelon material with strong unloading capacity) are shown in FIG. 2, and the results of testing sets of KASP-labeled primers (HEX-AGA2-X1, FAM-AGA2-Y1 and AGA2-C1) are consistent with those of the sets of KASP-labeled primers (HEX-AGA2-X2, FAM-AGA2-Y2 and AGA 2-C2). According to the judgment of the detection result, the accuracy rate of the method for detecting the material with strong unloading capacity of the raffinose in 115 natural population materials is 100%.
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
<110> agriculture and forestry academy of sciences of Beijing, Jing research and Yinong (Beijing) seed industry science and technology Co., Ltd
<120> SNP marker and KASP marker related to watermelon fruit raffinose unloading capability
<130> GNCFY192291
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 216
<212> DNA
<213> watermelon (Citrullus lanatus)
<400> 1
gccgtaattg tagaaaatta ggttttcttc taaagttcaa acccttrggt tggtgttrgt 60
gatgtccaat gaagagacaa tctcgatttt atcgtccgaa tttcttgttt ctaggagcta 120
aaacatttat ttggtatttt tgatattttg tctttatgct ttctgattgg ttgataatgt 180
ttataatctt gcgcagcaac tgtttgttgc tcaaca 216
<210> 2
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ggttttcttc taaagttcaa acccttg 27
<210> 3
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
taggttttct tctaaagttc aaaccctta 29
<210> 4
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
aatcgagatt gtctcttcat tggacatc 28
<210> 5
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gattgtctct tcattggaca tcacc 25
<210> 6
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
agattgtctc ttcattggac atcact 26
<210> 7
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gaaaattagg ttttcttcta aagttcaaac cc 32
Claims (10)
1. The application of the substance for detecting the polymorphism or genotype of the SNP marker CHR4-12223999 and/or the SNP marker CHR4-12224010 in identifying or assisting in identifying the raffinose unloading capacity of the watermelon fruit;
the SNP marker CHR4-12223999 is located at 12223999 th nucleotide on No. 4 chromosome of the watermelon genome, and is A or G;
the SNP marker CHR4-12224010 is located at 12224010 th nucleotide on No. 4 chromosome of the watermelon genome, and is A or G.
2. The application of the substance for detecting the polymorphism or genotype of the SNP marker CHR4-12223999 and/or the SNP marker CHR4-12224010 in watermelon breeding;
the SNP marker CHR4-12223999 is located at 12223999 th nucleotide on No. 4 chromosome of the watermelon genome, and is A or G;
the SNP marker CHR4-12224010 is located at 12224010 th nucleotide on No. 4 chromosome of the watermelon genome, and is A or G.
3. Use according to claim 2, characterized in that: the watermelon breeding is to cultivate watermelon varieties with strong cotton seed sugar unloading capability of watermelon fruits.
4. Use according to any one of claims 1 to 3, characterized in that: the substance for detecting the polymorphism or the genotype of the SNP marker CHR4-12223999 and/or the SNP marker CHR4-12224010 comprises a substance A for detecting the polymorphism or the genotype of the SNP marker CHR4-12223999 and/or a substance B for detecting the polymorphism or the genotype of the SNP marker CHR 4-12224010;
the substance A is a set of primers for amplifying watermelon genome DNA fragments including the SNP marker CHR 4-12223999;
the substance B is a set of primers for amplifying watermelon genome DNA fragments including the SNP marker CHR 4-12224010.
5. Use according to claim 4, characterized in that: the substance A comprises a compound for A1) or A2) or A3) as follows:
A1) the primer set consists of a single-stranded DNA molecule or a derivative thereof shown in a sequence 2 in a sequence table, a single-stranded DNA molecule or a derivative thereof shown in a sequence 3 in the sequence table and a single-stranded DNA molecule shown in a sequence 4 in the sequence table;
A2) PCR reagents containing a 1);
A3) a kit comprising a1) or a 2);
the substance B comprises B1) or B2) or B3) as follows:
B1) the primer set consists of a single-stranded DNA molecule or a derivative thereof shown in a sequence 5 in a sequence table, a single-stranded DNA molecule or a derivative thereof shown in a sequence 6 in the sequence table and a single-stranded DNA molecule shown in a sequence 7 in the sequence table;
B2) PCR reagents containing B1);
B3) a kit comprising B1) or B2).
6. Use according to claim 5, characterized in that: the derivative of the single-stranded DNA molecule shown in the sequence 2 in the sequence table is obtained by connecting the 5' end of the single-stranded DNA molecule shown in the sequence 2 with a fluorescent label A; the derivative of the single-stranded DNA molecule shown in the sequence 3 in the sequence table is obtained by connecting the 5' end of the single-stranded DNA molecule shown in the sequence 3 with a fluorescent label B; the fluorescent label A is different from the fluorescent label B;
the derivative of the single-stranded DNA molecule shown in the sequence 5 in the sequence table is obtained by connecting the 5' end of the single-stranded DNA molecule shown in the sequence 5 with a fluorescent label C; the derivative of the single-stranded DNA molecule shown in the sequence 6 in the sequence table is obtained by connecting the 5' end of the single-stranded DNA molecule shown in the sequence 6 with a fluorescent label D; the fluorescent label C is different from the fluorescent label D.
7. The method for identifying or assisting in identifying the raffinose unloading capacity of the watermelon fruit is characterized by comprising the step of identifying or assisting in identifying the raffinose unloading capacity of the watermelon fruit to be detected according to the genotype in order to detect the genotype of the SNP marker CHR4-12223999 and/or the SNP marker CHR4-12224010 in the genome of the watermelon to be detected.
8. A method of breeding a watermelon, comprising: detecting the genotype of the SNP marker CHR4-12223999 and/or the SNP marker CHR4-12224010 in the watermelon genome to be detected, and selecting the watermelon with the genotype of the SNP marker CHR4-12223999 and/or the genotype of the SNP marker CHR4-12224010 in the watermelon genome to be detected as GG for breeding;
the genotype of the SNP marker CHR4-12223999 in the watermelon genome to be detected is the homozygous type of the SNP marker CHR4-12223999 in the watermelon genome which is GG;
the genotype of the SNP marker CHR4-12224010 in the watermelon genome to be detected is GG which is homozygous for the SNP marker CHR4-12223999 in the watermelon genome which is G.
9. A method for detecting the genotype of an SNP marker CHR4-12223999 and/or an SNP marker CHR4-12224010 in a watermelon genome to be detected is A) or B):
A) direct sequencing;
B) carrying out PCR reaction on the watermelon genomic DNA to be detected by using the substance for detecting the genotype of the SNP marker CHR4-12223999 and/or the SNP marker CHR4-12224010 as set forth in any one of claims 1 to 4 to obtain a product, and carrying out genotyping on the product.
10. The method of claim 9, wherein:
the direct sequencing is to detect the nucleotide types of the SNP marker CHR4-12223999 and/or the SNP marker CHR4-12224010 in the watermelon genome to be detected: the SNP marker CHR4-12223999 is homozygous for A, and the genotype of the SNP marker CHR4-12223999 is AA; the SNP marker CHR4-12223999 is homozygous for G, and the genotype of the SNP marker CHR4-12223999 is GG; the SNP marker CHR4-12223999 is a hybrid type of A and G, and the genotype of the SNP marker CHR4-12223999 is AG;
the SNP marker CHR4-12224010 is homozygous for A, and the genotype of the SNP marker CHR4-12224010 is AA; the SNP marker CHR4-12224010 is homozygous for G, and the genotype of the SNP marker CHR4-12224010 is GG; the SNP marker CHR4-12224010 is a hybrid type of A and G, and the genotype of the SNP marker CHR4-12224010 is AG;
the genotyping method comprises detecting the fluorescent signal of the product, determining the genotype according to the fluorescent signal: if the product only shows the color of the fluorescent label connected to the 5' end of the DNA molecule shown in the sequence 2, the genotype of the SNP marker CHR4-12223999 in the watermelon genome to be detected is GG; if the product only shows the color of the fluorescent label connected to the 5' end of the DNA molecule shown in the sequence 3, the genotype of the SNP marker CHR4-12223999 in the watermelon genome to be detected is AA; if the product only shows the color of the DNA molecule 5 'end connected with the fluorescent label shown in the sequence 2 and the superimposed color of the DNA molecule 5' end connected with the fluorescent label shown in the sequence 3, the genotype of the SNP marker CHR4-12223999 in the watermelon genome to be detected is AG;
if the product only shows the color of the fluorescent label connected to the 5' end of the DNA molecule shown in the sequence 5, the genotype of the SNP marker CHR4-12224010 in the watermelon genome to be detected is GG; if the product only shows the color of the fluorescent label connected to the 5' end of the DNA molecule shown in the sequence 6, the genotype of the SNP marker CHR4-12224010 in the watermelon genome to be detected is AA; and if the product only shows the color of the DNA molecule 5 'end connected with the fluorescent label shown in the sequence 5 and the superimposed color of the DNA molecule 5' end connected with the fluorescent label shown in the sequence 6, the genotype of the SNP marker CHR4-12224010 in the watermelon genome to be detected is AG.
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| CN111961747A (en) * | 2020-09-10 | 2020-11-20 | 北京市农林科学院 | Method for assisting in identifying sugar content of watermelon fruit, SNP marker and KASP marker |
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