SNP (Single Nucleotide Polymorphism) molecular marker combination for bt type sweet corn and application of SNP molecular marker combination

Abstract

The invention provides a bt type sweet corn SNP molecular marker combination and application thereof, and belongs to the technical field of gene engineering. Comprising a first molecular marker and a second molecular marker; the nucleotide sequence of the first molecular marker is shown as SEQ ID No.1 or SEQ ID NO.8, the 264th base from the 5'end of the sequence is an SNP site, and the base is A or G; the nucleotide sequence of the second molecular marker is as shown in SEQ ID No.2 or SEQ ID NO.9, the 214th basic group of the sequence from the 5'end is an SNP site, and the basic group is T or G. Compared with a traditional bt type sweet corn selective breeding method, two SNP molecular markers are developed by researching and controlling genes of bt type sweet corn, and two pairs of primers capable of specifically amplifying the SNP markers are provided. Therefore, the sweet corn sample can be quickly, efficiently and accurately identified through PCR amplification and agarose gel electrophoresis without depending on experience of related breeders, and time and labor are saved.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a combination of SNP molecular markers for bt-type sweet corn and its application. Background Technology

[0002] Sweet corn is an important vegetable and grain crop, widely cultivated and consumed globally due to its sweet taste, rich nutrition, and good economic benefits. The sweetness type of sweet corn is controlled by different single-gene mutations, with Bt-type sweet corn being a significant category. Its sweetness phenotype is caused by a recessive mutation in the bt2 gene (Brittle-2) located on the short arm of chromosome 5. Compared to the traditional su1 type (normal sweetness), Bt-type sweet corn (often grouped with sh2 type as "super sweet") has significant commercial advantages such as high sugar content, long sweetness retention, and relatively tender pericarp, making it the mainstream type in the modern fresh and processed sweet corn market. However, traditional Bt-type sweet corn breeding heavily relies on field phenotypic selection, which has inherent drawbacks such as long cycles, high costs, low efficiency, and significant susceptibility to environmental interference, making phenotypic selection particularly difficult.

[0003] Therefore, developing efficient and precise molecular marker-assisted selection technology is key to achieving breakthroughs in Bt-type sweet corn breeding. Summary of the Invention

[0004] The purpose of this invention is to propose a combination of SNP molecular markers for bt-type sweet corn and its application, which can shorten the phenotypic selection cycle in bt-type sweet corn breeding, saving time and effort.

[0005] The technical solution of this invention is implemented as follows:

[0006] The technical solution adopted in this invention is:

[0007] In a first aspect, the present invention provides a bt-type sweet corn SNP molecular marker combination, comprising a first molecular marker and a second molecular marker;

[0008] The nucleotide sequence of the first molecular marker is shown in SEQ ID No. 1 or SEQ ID No. 8. The 264th base from the 5' end of the sequence is an SNP site, and its base is A or G.

[0009] The nucleotide sequence of the second molecular marker is shown in SEQ ID No. 2 or SEQ ID No. 9. The 214th base from the 5' end of the sequence is an SNP site, and its base is T or G.

[0010] This invention screened two SNP sites in the coding region of the maize bt2 gene (gene ID: Zm00001d050032). These two SNP sites are located at nucleotides 798 and 1175 of the bt2 gene coding region shown in SEQ ID No. 3, respectively. Based on these two SNP sites, this invention further developed and designed a first molecular marker and a second molecular marker. The first molecular marker having a base of G at the SNP site, or the second molecular marker having a base of G at the SNP site, or both SNP sites having bases of G, correspond to bt-type sweet maize. The two markers are used in combination for the detection and breeding of bt-type sweet maize.

[0011] In a second aspect, the present invention provides a primer pair for amplifying the above-mentioned SNP molecular marker pair, comprising: primer pair A for detecting a first molecular marker and primer pair B for detecting a second molecular marker.

[0012] The nucleotide sequences of primer pair A are shown in SEQ ID No. 4 and SEQ ID No. 5.

[0013] The nucleotide sequences of primer pair B are shown in SEQ ID No. 6 and SEQ ID No. 7.

[0014] A third aspect of the present invention provides the application of the above-described SNP molecular marker combination and primer pair in (1) or (2) below:

[0015] (1) Screening or identifying whether unknown sweet corn germplasm resources belong to the bt type sweet corn;

[0016] (2) Marker-assisted breeding of bt-type sweet corn, i.e. the selection and breeding of bt-type sweet corn inbred lines.

[0017] This invention offers the following advantages: Compared to traditional Bt-type sweet corn selection breeding methods, this invention, through research on the genes controlling Bt-type sweet corn, has developed two SNP molecular markers and provides two pairs of primers that can specifically amplify these SNP markers. Therefore, sweet corn samples can be rapidly and accurately identified using PCR amplification and agarose gel electrophoresis, without relying on the experience of relevant breeders, saving time and effort. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is the first molecular marker gel map of the present invention;

[0020] Figure 2 This is the second molecular marker gel map of the present invention;

[0021] Figure 3 This is a DNA sequence alignment diagram of the present invention;

[0022] Figure 4 This is a diagram showing the protein sequence alignment and secondary structure differences of the present invention. A: Amino acid sequence alignment results; B: Secondary structure of the protein encoded by the bt gene in common sweet corn; C: Secondary structure of the protein encoded by the bt gene in bt-type sweet corn. Detailed Implementation

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Example: A combination of two SNP molecular markers for identifying bt-type sweet corn

[0025] During the process of sweet corn breeding, the inventors selected three suspected bt-type sweet corn inbred lines based on traditional breeding experience. To clarify whether the sweet corn inbred lines were bt-type, the inventors designed a total of 7 pairs of primers containing all coding regions of the bt2 gene, based on the bt2 gene reference sequence provided in the NCBI database. After DNA extraction, PCR amplification, and sequencing of the three materials and ordinary corn materials, they found that single-base mutations appeared in the encoder sequences amplified by two pairs of primers. Based on this, two bt-type sweet corn SNP molecular markers were developed.

[0026] SNP molecular markers include a first molecular marker and a second molecular marker, wherein:

[0027] The nucleotide sequence of the first molecular marker is shown in SEQ ID No. 1 or SEQ ID No. 8. The 264th base from the 5' end of the sequence is an SNP site, and its base is A or G. In bt-type sweet corn, the nucleotide sequence with the 264th molecular base from the 5' end is G, as shown in SEQ ID No. 8.

[0028] The nucleotide sequence of the second molecular marker is shown in SEQ ID No. 2 or SEQ ID No. 9. The 214th base from the 5' end of the sequence is an SNP site, and its base is T or G. In bt-type sweet corn, the 264th molecular base from the 5' end of the nucleotide sequence is G, as shown in SEQ ID No. 9.

[0029] The PCR amplification primer sequences for the above-mentioned SEQ ID No. 1 sequence are: bt2-798-F and bt2-798-R; the PCR amplification primer sequences for the above-mentioned SEQ ID No. 2 sequence are: bt2-1175-F and bt2-1175-R. The specific sequences are as follows:

[0030] bt2-798-F:5'-GTACCCTGACATTTCATTTCTGAAAA-3'(SEQ ID No.4);

[0031] bt2-798-R:5'-TGCTACAGTAAAAGCACTGCTTACTTT-3' (SEQ ID No. 5);

[0032] bt2-1175-F:5'-GGGCTGCCACTATCCAGCA-3'(SEQ ID No.6);

[0033] bt2-1175-R:5'-GCATGTCGCACGTTCACCTG-3' (SEQ ID No. 7).

[0034] Eight samples were collected, consisting of two leaves each from common maize CML460 and suspected bt-type sweet maize inbred lines CP6Dbt, Gb002, and Gbs001. DNA was extracted from the samples using the CTAB method. PCR amplification was then performed using the sample DNA as a template, with primers bt2-798-F and bt2-798-R as the first molecular marker and bt2-1175-F and bt2-1175-R as the second molecular marker. The reaction system is shown in Table 1, and the amplification program is shown in Table 2. Successful amplification was determined by 1% agarose gel electrophoresis. After successful amplification, the sequences were sequenced and compared with a reference sequence.

[0035] Table 1: PCR reaction system

[0036] Table 2: PCR amplification program

[0037] The corresponding bt-type sweet corn is characterized by the base G at the SNP site of the first molecule marker, the base G at the SNP site of the second molecule marker, or both SNP sites containing the base G.

[0038] Based on this, the inventors used two pairs of markers to perform PCR testing on 256 materials from the F2 generation population generated by crossing an sh-type sweet corn inbred line as the male parent and a bt-type sweet corn inbred line as the female parent. The detection system and procedure were the same as those in Tables 1 and 2 above, and the PCR detection results are shown in Table 3.

[0039] Table 3: PCR test results of 256 samples

[0040] Material First molecular marker genotype Second molecular marker genotype Material First molecular marker genotype Second molecular marker genotype 25AF01 A T 25AF129 A T 25AF02 A T 25AF130 G T 25AF03 A T 25AF131 A T 25AF04 A T 25AF132 A G 25AF05 A T 25AF133 A T 25AF06 A T 25AF134 A T 25AF07 A T 25AF135 A G 25AF08 A T 25AF136 A T 25AF09 A T 25AF137 G G 25AF10 A T 25AF138 A T 25AF11 A T 25AF139 A T 25AF12 G T 25AF140 A G 25AF13 A G 25AF141 A T 25AF14 A T 25AF142 A T 25AF15 A T 25AF143 G T 25AF16 A T 25AF144 A T 25AF17 G T 25AF145 G G 25AF18 A T 25AF146 A T 25AF19 A T 25AF147 A T 25AF20 G T 25AF148 G T 25AF21 A T 25AF149 A T 25AF22 A T 25AF150 A T 25AF23 G T 25AF151 G T 25AF24 A T 25AF152 A G 25AF25 A G 25AF153 A T 25AF26 A T 25AF154 A T 25AF27 A T 25AF155 A T 25AF28 A G 25AF156 G T 25AF29 A T 25AF157 A G 25AF30 G T 25AF158 A T 25AF31 A G 25AF159 A T 25AF32 A T 25AF160 A G 25AF33 G G 25AF161 A G 25AF34 A T 25AF162 A T 25AF35 G T 25AF163 G T 25AF36 G G 25AF164 A T 25AF37 A T 25AF165 A G 25AF38 A G 25AF166 A T 25AF39 A T 25AF167 A T 25AF40 A T 25AF168 A G 25AF41 A T 25AF169 A T 25AF42 G T 25AF170 G T 25AF43 G G 25AF171 A T 25AF44 A T 25AF172 A T 25AF45 A T 25AF173 A G 25AF46 A G 25AF174 A G 25AF47 A G 25AF175 A T 25AF48 A T 25AF176 A T 25AF49 G G 25AF177 A T 25AF50 A T 25AF178 A G 25AF51 A T 25AF179 A T 25AF52 G T 25AF180 G T 25AF53 A T 25AF181 A T 25AF54 A G 25AF182 G T 25AF55 A T 25AF183 G G 25AF56 A T 25AF184 A T 25AF57 G T 25AF185 G T 25AF58 A T 25AF186 A T 25AF59 A T 25AF187 A T 25AF60 A G 25AF188 A G 25AF61 A T 25AF189 G T 25AF62 A T 25AF190 A T 25AF63 A G 25AF191 A T 25AF64 A T 25AF192 A G 25AF65 G G 25AF193 A T 25AF66 G T 25AF194 G G 25AF67 A T 25AF195 A T 25AF68 A T 25AF196 A G 25AF69 A G 25AF197 G T 25AF70 A T 25AF198 A T 25AF71 A T 25AF199 A T 25AF72 G T 25AF200 A T 25AF73 G T 25AF201 A T 25AF74 A T 25AF202 G G 25AF75 A G 25AF203 G T 25AF76 A T 25AF204 A T 25AF77 G T 25AF205 A T 25AF78 A T 25AF206 A G 25AF79 A T 25AF207 A T 25AF80 A T 25AF208 G T 25AF81 G T 25AF209 A T 25AF82 A T 25AF210 G G 25AF83 A T 25AF211 G G 25AF84 G T 25AF212 G T 25AF85 A T 25AF213 A T 25AF86 A G 25AF214 A T 25AF87 A G 25AF215 G T 25AF88 A T 25AF216 A T 25AF89 G T 25AF217 G T 25AF90 A T 25AF218 A T 25AF91 A G 25AF219 A G 25AF92 A T 25AF220 A T 25AF93 G T 25AF221 A T 25AF94 A T 25AF222 A T 25AF95 G T 25AF223 G T 25AF96 A G 25AF224 A T 25AF97 A T 25AF225 A G 25AF98 G T 25AF226 G T 25AF99 A T 25AF227 A G 25AF100 A T 25AF228 A T 25AF101 A T 25AF229 A G 25AF102 G T 25AF230 A T 25AF103 A T 25AF231 A T 25AF104 A G 25AF232 A T 25AF105 A T 25AF233 G T 25AF106 A G 25AF234 A T 25AF107 A G 25AF235 G T 25AF108 A T 25AF236 A T 25AF109 A G 25AF237 A G 25AF110 A T 25AF238 A T 25AF111 G T 25AF239 A T 25AF112 A G 25AF240 A T 25AF113 A T 25AF241 G T 25AF114 A T 25AF242 A T 25AF115 A T 25AF243 A T 25AF116 A G 25AF244 G T 25AF117 G T 25AF245 A T 25AF118 A T 25AF246 A T 25AF119 A G 25AF247 A G 25AF120 G G 25AF248 A T 25AF121 A T 25AF249 G T 25AF122 A G 25AF250 A T 25AF123 A T 25AF251 A G 25AF124 G T 25AF252 A T 25AF125 A T 25AF253 G G 25AF126 A G 25AF254 A T 25AF127 G G 25AF255 A T 25AF128 A T 25AF256 G T

[0041] As shown in Table 3, there were 111 bt-type sweet corn materials in the F2 generation, with a total of three genotypes. Among them, 48 materials had the first molecular marker genotype A and the second molecular marker genotype G; 48 materials had the first molecular marker genotype G and the second molecular marker genotype T; and 15 materials had the first molecular marker genotype G and the second molecular marker genotype G.

[0042] Compared with traditional bt-type sweet corn breeding, this invention uses two SNP molecular markers to rapidly identify sweet corn breeding materials. It uses two pairs of primers to perform PCR amplification on the sample genomic DNA and directly obtains the identification results without relying on the field selection experience of breeders, thus saving breeding time and effort.

[0043] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A bt-type sweet corn SNP molecular marker combinatorial, characterized in that, Including first molecular markers and second molecular markers; The nucleotide sequence of the first molecular marker is shown in SEQ ID No. 1 or SEQ ID No.

8. The 264th base from the 5' end of the sequence is an SNP site, and its base is A or G. The nucleotide sequence of the second molecular marker is shown in SEQ ID No. 2 or SEQ ID No.

9. The 214th base from the 5' end of the sequence is an SNP site, and its base is T or G.

2. A primer combination for amplifying the above-mentioned SNP molecular marker combination, characterized in that, include: Primer pair A for detecting the first molecular marker and primer pair B for detecting the second molecular marker; the nucleotide sequences of primer pair A are shown in SEQ ID No. 4 and SEQ ID No. 5; the nucleotide sequences of primer pair B are shown in SEQ ID No. 6 and SEQ ID No.

7.

3. The application of the SNP molecular marker combination as described in claim 1 or the primer combination as described in claim 2, characterized in that, The applications include: (1) Screening or identifying whether unknown sweet corn germplasm resources belong to the Bt type sweet corn; (2) Marker-assisted breeding of bt-type sweet corn, i.e. the selection and breeding of bt-type sweet corn inbred lines.

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