A SNP molecular marker for identifying buckwheat husk thickness, a KASP primer set, and its application

Abstract

The present invention relates to the technical field of DNA molecular markers, and in particular to a single-nucleotide polymorphism (SNP) molecular marker for identifying buckwheat husk thickness, a KASP primer set, and applications thereof. The present invention provides a single-nucleotide polymorphism (SNP) molecular marker for identifying buckwheat husk thickness. The SNP molecular marker is located at nucleotide position 6815573 of chromosome 1 of buckwheat, and the mutant base is C or T. The SNP molecular marker can be used to predict, identify, and screen for the trait of buckwheat husk thickness.

Description

Technical Field

[0001] The present invention relates to the technical field of DNA molecular markers, and in particular to a SNP molecular marker for identifying the thickness of tartary buckwheat husk, a KASP primer set and applications thereof. Background Art

[0002] Tartary buckwheat (Fagopyrum tataricum (L.) Gaertn.), a member of the Polygonaceae family, is a traditional coarse grain known as a "three-reduction food" and a food and medicine crop. Conventionally cultivated buckwheat husks (commonly known as buckwheat husks) are thick, typically ranging from 0.16 to 0.30 mm in thickness, with a hull ratio of 20 to 30%. This makes hulling difficult, requiring a series of steps including soaking, steaming, drying, and shelling. This makes it difficult to produce fully nutritious buckwheat rice with a bran layer high in rutin. This results in a loss of nutrients and medicinal ingredients, changes in product color, and increased processing costs. Furthermore, during direct milling, a small amount of hull dust can enter the food, reducing its palatability. This limits the processing of buckwheat products and significantly impacts the development of the buckwheat industry. The thin-shelled buckwheat husk mutant has a husk thickness generally between 0.04 and 0.14 mm, a shell ratio of 10 to 20%, and cracks at the husk grooves, making it easy to peel off and hull. However, due to its small grain size, low yield, and poor adaptability, it has not been widely planted. Therefore, introducing the thin-shell trait into ordinary thick-shelled buckwheat varieties and cultivating new thin-shelled, high-yield buckwheat varieties is one of the main breeding goals.

[0003] Because thick and thin tartary buckwheat husks are difficult to distinguish visually, especially when the husks are not removed, the two are often confused. Therefore, conventional methods for distinguishing tartary buckwheat husk thickness are relatively difficult. Modern DNA molecular marker technology provides an efficient method for identifying or early predicting tartary buckwheat husk thickness. Using specific DNA molecular markers associated with husk thickness, it is possible to identify tartary buckwheat thickness in a short period of time, thereby improving breeding efficiency. Therefore, establishing an efficient DNA molecular marker and its detection method has become one of the most urgent issues in identifying tartary buckwheat husk thickness and is of great significance to tartary buckwheat breeding. Summary of the Invention

[0004] The purpose of the present invention is to provide a SNP molecular marker for identifying the thickness of tartary buckwheat husk, a KASP primer set and an application thereof. The SNP molecular marker can be used to predict, identify and screen the tartary buckwheat husk thickness trait.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a SNP molecular marker for identifying the thickness of tartary buckwheat husk. The SNP molecular marker is located at the 6815573rd nucleotide of chromosome 1 of tartary buckwheat, and the mutant base is C or T.

[0007] Preferably, when the mutated base is C, the genotype is CC or CT;

[0008] When the mutant base is T, the genotype is TT;

[0009] The husk of Tartary buckwheat with genotype CC or CT is thick;

[0010] The husk of tartary buckwheat with genotype TT is thin.

[0011] The present invention also provides a KASP primer set for amplifying the SNP molecular marker. The forward primers of the KASP primer set are shown in SEQ ID NOs: 1 to 2; the reverse primers of the KASP primer set are shown in SEQ ID NO: 3.

[0012] The present invention also provides application of the KASP primer set in identifying the thickness of tartary buckwheat husk.

[0013] The present invention also provides the use of the KASP primer set in tartary buckwheat molecular marker-assisted breeding.

[0014] The present invention also provides a method for identifying the thickness of tartary buckwheat husk, comprising the following steps:

[0015] (1) using buckwheat DNA as a template and using the KASP primer set to amplify to obtain a PCR product;

[0016] (2) According to the results of PCR products, the thickness type of buckwheat husk was determined.

[0017] The present invention also provides a kit for identifying the thickness of tartary buckwheat husks, which comprises the KASP primer set.

[0018] The present invention also provides a kit for tartary buckwheat molecular marker-assisted breeding, which comprises the KASP primer set.

[0019] Beneficial effects of the present invention:

[0020] The present invention provides a single-nucleotide polymorphism (SNP) molecular marker for identifying buckwheat husk thickness. The SNP marker is located at nucleotide position 6815573 on chromosome 1 of buckwheat, where the mutated base is either C or T. The 100 base pairs before and after nucleotide position 6815573 are selected as PCR amplification sequences, and primers are designed for identification of buckwheat husks. This identification method is time-efficient and highly accurate, and can detect SNP mutation sites using a small amount of DNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Thick-shelled buckwheat and thin-shelled buckwheat. The left picture shows thick-shelled buckwheat, and the right picture shows thin-shelled buckwheat.

[0022] Figure 2 The following is the typing situation of 191 tartary buckwheat plants with different genotypes;

[0023] Figure 3 The typing results of 156 tartary buckwheat plants with different genotypes are shown;

[0024] Figure 4 This is the classification of 15 different varieties of Tartary Buckwheat. DETAILED DESCRIPTION

[0025] The invention provides a SNP molecular marker for identifying the thickness of tartary buckwheat husk. The SNP molecular marker is located at the 6815573rd nucleotide of tartary buckwheat chromosome 1 (Genbank: GCA002319775.1), and the mutant base is C or T.

[0026] In the present invention, when the mutated base is C, the genotype is CC or CT;

[0027] When the mutant base is T, the genotype is TT;

[0028] The husk of Tartary buckwheat with genotype CC or CT is thick;

[0029] The husk of tartary buckwheat with genotype TT is thin.

[0030] The present invention also provides a KASP primer set for amplifying the SNP molecular marker, wherein the forward primers of the KASP primer set are shown in SEQ ID NOs: 1 to 2; the reverse primers of the KASP primer set are shown in SEQ ID NO: 3;

[0031] The SEQ ID NO: 1 is GAAGGTGACCAAGTTCATGCTATTGTTGTTGTGCAGTTCGATCTCC;

[0032] The SEQ ID NO: 2 is GAAGGTCGGAGTCAACGGATTATTGTTGTTGTGCAGTTCGATCTCT;

[0033] The SEQ ID NO: 3 is TCCAACACAGTTTGGTTTTCTCATTTGCC;

[0034] The 5' end of the forward primer shown in SEQ ID NO: 1 is connected to a fluorescent molecule FAM, which is used to amplify the DNA sequence of thick-shelled tartary buckwheat; the 5' end of the forward primer shown in SEQ ID NO: 2 is connected to a fluorescent molecule HEM, which is used to amplify the DNA sequence of thin-shelled tartary buckwheat;

[0035] The DNA sequence amplified using SEQ ID NO: 1 and SEQ ID NO: 3 is: ATTACTTTCAAATCATCAAATCTCATTTATGCTTATTATTTTTCAACCAAGGCAAAACCTTTGCACGCAGAAACTGATTGTTGTTGTGCAGTTCGATCTCCTGCAAAAGGCAAATGAGAAAACCAAACTGTGTTGGATTTCGGACAGACCAAAATCTTGCTTGCAATATAGTAGGTATAGCTTTTAAGGAAATTAGCAATTTCTGGCATGTCACATTTTATCATCC (SEQ ID NO: 4), where the position of the mutated base is base 101 in the sequence of SEQ ID NO: 4;

[0036] The DNA sequence amplified using SEQ ID NO: 2 and SEQ ID NO: 3 was: ATTACTTTCAAATCATCAAATCTCATTTATGCTTATTATTTTTCAACCAAGGCAAAACCTTTGCACGCAGAAACTGATTGTTGTTGTGCAGTTCGATCTCTTGCAAAAGGCAAATGAGAAAACCAAACTGTGTTGGATTTCGGACAGACCAAAATCTTGCTTGCAATATAGTAGGTATAGCTTTTAAGGAAATTAGCAATTTCTGGCATGTCACATTTTATCATCC (SEQ ID NO: 5), and the position of the mutated base was base 101 in the sequence of SEQ ID NO: 5.

[0037] The present invention also provides application of the KASP primer set in identifying the thickness of tartary buckwheat husk.

[0038] The present invention also provides the use of the KASP primer set in tartary buckwheat molecular marker-assisted breeding.

[0039] The present invention also provides a method for identifying the thickness of tartary buckwheat husk, comprising the following steps:

[0040] (1) using buckwheat DNA as a template and using the KASP primer set to amplify to obtain a PCR product;

[0041] (2) According to the results of PCR products, the thickness type of buckwheat husk was determined.

[0042] In the present invention, when amplifying the DNA of tartary buckwheat, 100 bp before and after the 6815573rd nucleotide are selected as the amplified sequence;

[0043] The preparation method of the KASP primer set comprises the following steps: dissolving primers SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 to 50 μM in a Tris-EDTA buffer solution (pH 8.0), and then mixing them in a volume ratio of SEQ ID NO: 1: SEQ ID NO: 2: SEQ ID NO: 3 = 1:1:3 to obtain the KASP primer set;

[0044] The amplification system is as follows: 5 μL template, 5 μL FLU-ARMS for KASP 2×PCR mix, 0.125 μL KASP primer set;

[0045] The concentration of DNA in the template is 2.5-3 ng / μL, preferably 2.75 ng / μL;

[0046] The amplification conditions are as follows: 95°C for 10 min; 95°C for 15 s, 61°C to 55°C for 45 s, 10 cycles, with the temperature decreasing by 0.6°C each cycle; 95°C for 15 s, 55°C for 45 s, 36 cycles; 30°C for 30 s;

[0047] The fluorescence results were automatically analyzed on an ABI 7900HT fluorescence quantitative PCR instrument using endpoint fluorescence scanning;

[0048] If the result shows blue fluorescence, the tartary buckwheat husk is a homozygous thick husk; if the result shows red fluorescence, the tartary buckwheat husk is a homozygous thin husk; if the result shows blue fluorescence and red fluorescence, the tartary buckwheat husk is a heterozygous thick husk.

[0049] The present invention also provides a kit for identifying the thickness of tartary buckwheat husks, which comprises the KASP primer set.

[0050] The present invention also provides a kit for tartary buckwheat molecular marker-assisted breeding, which comprises the KASP primer set.

[0051] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0052] Example 1

[0053] (1) KASP primer set: Primers were designed based on the 100 bp before and after nucleotide 6815573 of Tartary Buckwheat chromosome 1, as shown in Table 1 , and were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0054] Table 1 SNP typing primer sequence information

[0055]

[0056] Note: The 5' end of SEQ ID NO: 1 is connected to the fluorescent molecule FAM; the 5' end of SEQ ID NO: 2 is connected to the fluorescent molecule HEM;

[0057] Primers SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 were dissolved in Tris-EDTA buffer (pH 8.0) to 50 μM, and then mixed in a volume ratio of SEQ ID NO: 1: SEQ ID NO: 2: SEQ ID NO: 3 = 1:1:3 to obtain a KASP primer set.

[0058] (2) Extraction of DNA from buckwheat leaves

[0059] A. Take 200 mg of leaves from a single buckwheat plant, grind them evenly with liquid nitrogen, and place them in a 2 mL EP tube to obtain the sample to be tested.

[0060] B. Use DNA UN1Q-10 column extraction kit (purchased from Thermo Fisher Scientific) to extract the DNA of the sample to be tested to obtain the DNA of the buckwheat to be tested;

[0061] C. Detect DNA using 1.5% agarose gel electrophoresis at a DNA concentration of 200-300 ng / μL;

[0062] (3) using the DNA of the buckwheat to be tested in step (2)B as a template, and using the KASP primer set in step (1) to amplify to obtain a PCR product;

[0063] The amplification system was as follows: 5 μL DNA template, 5 μL FLU-ARMS for KASP 2× PCR mix, and 0.125 μL KASP primer set. The final concentration of the DNA template in the system was 3 ng / μL.

[0064] The amplification conditions were as follows: pre-denaturation (95°C for 10 min), touchdown PCR for 10 cycles (95°C for 15 s, 61°C to 55°C for 45 s, with a temperature drop of 0.6°C per cycle), amplification for 36 cycles (95°C for 15 s, 55°C for 45 s), and plate reading (30°C for 30 s).

[0065] The DNA sequence amplified using SEQ ID NO: 1 and SEQ ID NO: 3 is: ATTACTTTCAAATCATCAAATCTCATTTATGCTTATTATTTTTCAACCAAGGCAAAACCTTTGCACGCAGAAACTGATTGTTGTTGTGCAGTTCGATCTCCTGCAAAAGGCAAATGAGAAAACCAAACTGTGTTGGATTTCGGACAGACCAAAATCTTGCTTGCAATATAGTAGGTATAGCTTTTAAGGAAATTAGCAATTTCTGGCATGTCACATTTTATCATCC (SEQ ID NO: 4), where the position of the mutated base is base 101 in the sequence of SEQ ID NO: 4;

[0066] The DNA sequence amplified using SEQ ID NO: 2 and SEQ ID NO: 3 is: ATTACTTTCAAATCATCAAATCTCATTTATGCTTATTATTTTTCAACCAAGGCAAAACCTTTGCACGCAGAAACTGATTGTTGTTGTGCAGTTCGATCTCTTGCAAAAGGCAAATGAGAAAACCAAACTGTGTTGGATTTCGGACAGACCAAAATCTTGCTTGCAATATAGTAGGTATAGCTTTTAAGGAAATTAGCAATTTCTGGCATGTCACATTTTATCATCC (SEQ ID NO: 5), where the position of the mutated base is base 101 in the sequence of SEQ ID NO: 5;

[0067] The fluorescence results were automatically analyzed on an ABI 7900HT fluorescence quantitative PCR instrument using endpoint fluorescence scanning. The blue fluorescence indicated homozygous thick-shelled fruit, the red fluorescence indicated homozygous thin-shelled fruit, and the heterozygous thick-shelled fruit showed both blue and red fluorescence.

[0068] Thick shell buckwheat and thin shell buckwheat Figure 1 shown.

[0069] Example 2

[0070] The method described in Example 1 was used to test 191 individual plants of the F2 population of the thick-shelled Yunqiao No. 1 and the thin-shelled Xiaoxiaoqiao, and a negative control (not containing any sample DNA) was set up. The test results are shown in Tables 2 and Figure 2 shown.

[0071] Table 2. Typing results of 191 individual strains in the F2 population

[0072]

[0073] From Table 2 and Figure 2 It can be seen that there are 15 individual plants whose bases could not be determined after the second test, including 8 plants with homozygous thick fruit shells, 4 plants with heterozygous thick fruit shells, 3 plants with homozygous thin fruit shells, and no mutant bases. The success rate is 92.15%.

[0074] Example 3

[0075] The method described in Example 1 was used to test 156 individual plants of the F3 population of the thick-shelled Yunqiao No. 1 and the thin-shelled Xiaoxiaoqiao, and a negative control (not containing any sample DNA) was set up. The test results are shown in Tables 3 and Figure 3 shown.

[0076] Table 3. Typing results of 156 individual strains in the F3 population

[0077]

[0078] From Table 3 and Figure 3 It can be seen that there are 5 individual plants whose bases could not be determined after the second test, including 2 homozygous thick fruit shells, 2 heterozygous thick fruit shells, and 1 homozygous thin fruit shell. One homozygous thin fruit shell plant had a base mutation, and the success rate was 96.15%.

[0079] Example 4

[0080] Fifteen different varieties of buckwheat were tested using the method described in Example 1. Two strains of each type of buckwheat were tested in parallel. The variety types are shown in Table 4. A negative control (not containing any sample DNA) was set up. The test results are shown in Tables 5 and Figure 4 As shown;

[0081] Table 4 Classification of 15 Tartary Buckwheat varieties

[0082]

[0083]

[0084] Table 5 Loci typing of 15 tartary buckwheat varieties

[0085]

[0086] From Table 5 and Figure 4 It can be seen that the number of homozygous thick fruit shells is 26, the number of homozygous thin fruit shells is 4, there is no mutation and uncertain base, and the success rate of homozygous thick fruit shells and homozygous thin fruit shells is 100%.

[0087] As can be seen from the above examples, the present invention provides a SNP molecular marker for identifying tartary buckwheat husk thickness, a KASP primer set and applications thereof. The SNP molecular marker can be used to predict, identify and screen the tartary buckwheat husk thickness trait.

[0088] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Application of a reagent for identifying SNP molecular markers in identifying the thickness of tartary buckwheat husks, characterized in that: The SNP molecular marker is located at nucleotide 6815573 of chromosome 1 of buckwheat, with a Genbank ID of GCA002319775.1, and the mutant base is C or T; When the mutated base is C, the genotype is CC or CT; When the mutant base is T, the genotype is TT; The husk of Tartary buckwheat with genotype CC or CT is thick; The husk of tartary buckwheat with genotype TT is thin.

2. The use according to claim 1, characterized in that The KASP primer set for amplifying 100 bp before and after the SNP molecular marker includes a forward primer and a reverse primer; the forward primer is shown in SEQ ID NO: 1-2; the reverse primer is shown in SEQ ID NO:

3.

3. A method for identifying the thickness of tartary buckwheat husk, characterized in that: The steps include: (1) Using buckwheat DNA as a template, the KASP primer set was used for amplification to obtain a PCR product; (2) Determine the thickness type of tartary buckwheat shell based on the results of PCR products; The KASP primer set amplifies the SNP molecular marker; The SNP molecular marker is located at the 6815573rd nucleotide of chromosome 1 of tartary buckwheat, with a Genbank ID of GCA002319775.1, and the mutant base is C or T.

4. The method according to claim 3, characterized in that The KASP primer set includes a forward primer and a reverse primer; the forward primer is shown in SEQ ID NO: 1-2; the reverse primer is shown in SEQ ID NO: 3.

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