CN108504767B - Soybean molecular marker and application thereof in detecting nutritive value of soybean seeds - Google Patents
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Abstract
The invention discloses a soybean molecular marker and application thereof in detecting the nutritive value of soybean seeds. The disclosed soybean molecular markers of the invention are A1) or A2): A1) using soybean genome DNA as a template, and adopting a primer pair A1 to amplify to obtain a DNA molecule; the A1 consists of two single-stranded DNAs with the names of A1-F and A1-R respectively, the A1-F is the single-stranded DNA which is specifically combined with the position 346 upstream of the sequence 1 in the soybean genome, and the A1-R is the single-stranded DNA which is specifically combined with the position 346 downstream of the sequence 1 in the soybean genome; A2) the soybean genome DNA has a nucleotide corresponding to the 346 th site of the sequence 1 in the sequence table. Experiments prove that the soybean molecular marker is related to the 11S/7S ratio in the seed protein, and the 11S/7S ratio in the seed protein can be detected by detecting the soybean molecular marker and the corresponding genotype.
Description
Technical Field
The invention relates to a soybean molecular marker and application thereof in detecting the nutritive value of soybean seeds in the field of biotechnology.
Background
The soybean seed protein is an important vegetable protein source and has rich nutritional value. Soybean is the fourth crop of China second only to corn, wheat and rice, and is one of the main food and oil crops in China. Due to its extremely high nutritional value, high physiological activity and wide industrial use, there is an increasing worldwide demand for soybeans.
70% of the storage proteins in soybean seeds are globulin and beta-conglobulin. Globulin and beta-conglobulin, which may also be referred to as 11S globulin and 7S globulin, are in turn significantly inversely related. The sulfur-containing amino acid content of the 7S globulin is 3-4 times lower than that of the 11S globulin, the nutritional value of the soybean is closely related to the content of the sulfur-containing amino acid (methionine and cysteine), and the higher the content of the sulfur-containing amino acid is, the higher the nutritional value is. Therefore, the research on the ratio of the 11S globulin to the 7S globulin and the content of sulfur-containing amino acid has important value for improving the nutritional value of the soybeans.
Disclosure of Invention
The invention aims to solve the technical problems of how to detect the nutritional value of soybean seeds, particularly the content of sulfur-containing amino acid, and how to detect the content of 11S globulin and 7S globulin in the soybean seeds and the ratio of the two.
In order to solve the technical problems, the invention firstly provides a soybean molecular marker, and the soybean molecular marker is A1) or A2):
A1) using soybean genome DNA as a template, and adopting a primer pair A1 to amplify to obtain a DNA molecule; the A1 consists of two single-stranded DNAs with the names of A1-F and A1-R respectively, the A1-F is the single-stranded DNA which is specifically combined with the position 346 upstream of the sequence 1 in the soybean genome, and the A1-R is the single-stranded DNA which is specifically combined with the position 346 downstream of the sequence 1 in the soybean genome;
A2) the soybean genome DNA has a nucleotide corresponding to the 346 th site of the sequence 1 in the sequence table. The polymorphism of the soybean molecular marker can be G or A at the 346 th position corresponding to the sequence 1 in the soybean genome.
The A1-F can be single-stranded DNA shown in the 1 st-20 th position of the sequence 1, and the A1-R can be single-stranded DNA reverse complementary to the 1578 th-1598 th position of the sequence 1.
In order to solve the technical problems, the invention also provides a method for identifying the soybean genotype, wherein the genotypes comprise GG genotype, GA genotype and AA genotype, and the method comprises the following I or II or III or IV:
i, detecting 346 th nucleotide corresponding to a sequence 1 in a sequence table in a soybean genome to be detected, wherein the soybean to be detected is a GG genotype if two chromosomes in the soybean genome to be detected are g 1); if the two chromosomes in the genome of the soybean to be detected are the chromosomes of g2), the soybean to be detected is an AA genotype; if one of the two chromosomes in the genome of the soybean to be detected is the chromosome of g1) below and the other chromosome is the chromosome of g2) below, the soybean to be detected is GA genotype;
g1) the nucleotide corresponding to 346 th site of a sequence 1 in a sequence table is G;
g2) the nucleotide corresponding to 346 th site of a sequence 1 in a sequence table is A;
II, the following K1) and K2):
K1) taking the soybean genome DNA to be detected as a template, and performing PCR amplification by adopting the A1 to obtain a PCR product;
K2) detecting the sequence of the PCR product obtained in the step K1), and determining the soybean genotype according to the PCR product:
the genotype of the PCR product corresponding to the soybean to be detected with G at the 346 th site of the sequence 1 is GG genotype; the genotype of the PCR product corresponding to the soybean to be detected with G and A at the 346 th site of the sequence 1 is GA genotype; the genotype of the PCR product corresponding to the soybean to be detected, of which the 346 th site of the sequence 1 is A, is an AA genotype;
iii, L1) and L2) as follows:
l1) taking the DNA of the soybean genome to be detected as a template, and carrying out PCR amplification by adopting a primer pair named A2 to obtain a PCR product; the A2 consists of two single-stranded DNAs with the names of A2-F and A2-R respectively, and the sequences of the A2-F and the A2-R are the sequences 3 and 4 in a sequence table respectively;
l2), determining the soybean genotype from the PCR product: the genotype of the PCR product corresponding to the soybean to be detected with G at the 346 th site of the sequence 1 is GG genotype; the genotype of the PCR product corresponding to the soybean to be detected with G and A at the 346 th site of the sequence 1 is GA genotype; the genotype of the PCR product corresponding to the soybean to be detected, of which the 346 th site of the sequence 1 is A, is an AA genotype;
iv, M1) and M2) as follows:
m1) taking the soybean genome DNA to be detected as a template, carrying out PCR amplification by adopting the A2 to obtain a PCR product, and carrying out enzyme digestion on the PCR product by utilizing a restriction enzyme Taq I to obtain an enzyme digestion product;
m2), detecting the size of the enzyme cutting product, and determining the soybean genotype according to the size of the enzyme cutting product: if the enzyme digestion product contains a strip with the size of 158bp, the soybean to be detected is GG genotype soybean, if the enzyme digestion product contains strips with the sizes of 158bp, 20 bp and 138bp, respectively, the soybean to be detected is GA genotype soybean, if the enzyme digestion product contains strips with the sizes of 20 bp and 138bp, the soybean to be detected is AA genotype soybean.
The PCR amplification system using the A1 can be as follows: DNA template, 4. mu.l (30 ng/. mu.l); KOD-Plus-Neo, 0.4. mu.l; 10 × Buffer, 3.0 μ l; 2.0mM dNTPs, 3.0. mu.l; 25mM) MgSO (MgSO)4，1.8μl；10μM A1-F，1.0μl；10μM A1-R，1.0μl；ddH2O, 15.8 μ l; the total volume was 30. mu.l. Wherein KOD-Plus-Neo and 10 xBuffer can be products of Beijing Bailingke biotechnology Limited liability company respectively.
The PCR reaction program for PCR amplification by using the A1 can be as follows: firstly, 94 ℃ for 5 min; then, carrying out 36 cycles of 94 ℃ 30S, 55 ℃ 30S and 72 ℃ 30S; finally, extension is carried out for 10min at 72 ℃, and heat preservation is carried out at 4 ℃.
The PCR amplification system using the A2 can be as follows: DNA template 4. mu.l (20 ng/. mu.l); easy Taq DNA Polymerase 0.3. mu.l; 10 × Easy Taq Buffer 3 μ l; 2.5mM dNTPs 2.5. mu.l; A2-F3.0. mu.l (2. mu.M); A2-R3.0. mu.l (2. mu.M); ddH2Make up to 30. mu.l of O. Wherein, Easy Taq DNA Polymerase enzyme and 10 × Easy Taq Buffer are all products of Beijing Quanji Biotech Limited.
The reaction procedure for PCR amplification using the A2 can be: firstly, 94 ℃ for 5 min; then, carrying out 36 cycles of 94 ℃ 30S, 55 ℃ 30S and 72 ℃ 30S; finally, extension is carried out for 10min at 72 ℃, and heat preservation is carried out at 4 ℃.
In order to solve the technical problems, the invention also provides a method for detecting the nutritive value of soybean seeds, which comprises the following steps: detecting the genotype of the soybean to be detected by using the method for identifying the genotype of the soybean, wherein the nutritive value of GG genotype soybean seeds is lower than or candidate for being lower than that of GA genotype soybean; the nutritive value of GG genotype soybean seeds is lower than that of AA genotype soybean or candidate soybean seeds are lower than that of AA genotype soybean; the nutritional value of GA genotype soybean seeds is lower or the candidate is lower than that of AA genotype soybean.
In the above method, the nutritional value of the soybean seed may be represented by a1), a2), a3) or a 4):
a1) the content of the sulfur-containing amino acid in the soybean seeds;
a2) the ratio of 11S globulin to 7S globulin content in the soybean seed protein;
a3) 11S globulin content in soybean seed protein;
a4) the 7S globulin content of the soybean seed protein.
The higher the methionine content, the higher the nutritional value of the soybean seed, and the lower the methionine content, the lower the nutritional value of the soybean seed. The content of the sulfur-containing amino acid of GG genotype soybean seeds is lower than or lower than that of candidate GA genotype soybean; the content of the sulfur-containing amino acid of the GG genotype soybean seeds is lower than or lower than that of AA genotype soybean candidates; the GA genotype soybean seeds have a lower or candidate lower content of methionine than AA genotype soybeans.
The content of the methionine may also be expressed in the ratio of the content of 11S globulin to 7S globulin in the soybean seed protein. The ratio of the 11S globulin content to the 7S globulin content in the soybean seed protein was recorded as the 11S/7S ratio. The higher the 11S/7S ratio, the higher the content of the methionine in the soybean seeds, and the lower the 11S/7S ratio, the lower the content of the methionine in the soybean seeds; the higher the 11S globulin content in the soybean seed protein is, the higher the content of the methionine is, and the lower the 11S globulin content in the soybean seed protein is, the lower the content of the methionine is; the higher the 7S globulin content in the soybean seed protein, the lower the content of the methionine, and the lower the 7S globulin content in the soybean seed protein, the higher the content of the methionine. The higher the 11S/7S ratio, the higher the nutritional value of the soybean seeds, and the lower the 11S/7S ratio, the lower the nutritional value of the soybean seeds; the higher the 11S globulin content in the soybean seed protein, the higher the nutritional value, and the lower the 11S globulin content in the soybean seed protein, the lower the nutritional value; the higher the 7S globulin content in the soybean seed protein, the lower the nutritional value, and the lower the 7S globulin content in the soybean seed protein, the higher the nutritional value.
The 11S/7S ratio of GG genotype soybean seeds is lower than or lower than that of GA genotype soybean candidates; the 11S/7S ratio of GG genotype soybean seeds is lower than or lower than AA genotype soybean in a candidate way; the 11S/7S ratio of GA genotype soybean seeds is lower than or lower than that of AA genotype soybean candidates.
In order to solve the technical problem, the invention also provides any one of the following products:
H1) the A1;
H2) the A2;
H3) the kit consists of the A2 and a restriction enzyme Taq I.
The product can be used in any one of I1) -I5):
I1) detecting the soybean genotype;
I2) detecting the nutritive value of soybean seeds;
I3) detecting the content of the methionine in the soybean seeds;
I4) detecting the content of 11S globulin and/or 7S globulin in soybean seeds;
I5) and detecting the content ratio of 11S globulin to 7S globulin in the soybean seeds.
In order to solve the technical problem, the invention also provides any one of the following applications:
x1) application of the soybean molecular marker in detecting the nutritional value of soybean seeds;
x2) application of the soybean molecular marker in detecting the content of the methionine in soybean seeds;
x3) application of the soybean molecular marker in detecting the content of 11S globulin and/or 7S globulin in soybean seeds;
x4) application of the soybean molecular marker in detecting the content ratio of 11S globulin and 7S globulin in soybean seeds;
x5) the application of the soybean molecular marker in soybean breeding;
x6) application of the method for identifying the soybean genotype in detecting the nutritional value of soybean seeds;
x7) application of the method for identifying the soybean genotype in preparing products for detecting the nutritional value of soybean seeds;
x8) application of the method for identifying the soybean genotype in detecting the content of the methionine in the soybean seeds;
x9) application of the method for identifying the soybean genotype in preparing products for detecting the content of the methionine in soybean seeds;
x10) application of the method for identifying soybean genotype in detecting 11S globulin and/or 7S globulin content in soybean seeds;
x11) application of the method for identifying soybean genotype in preparation of products for detecting 11S globulin and/or 7S globulin content in soybean seeds;
x12) application of the method for identifying soybean genotype in detecting the content ratio of 11S globulin and 7S globulin in soybean seeds;
x13) application of the method for identifying soybean genotype in preparation of products for detecting content ratio of 11S globulin and 7S globulin in soybean seeds;
x14) the use of the method for identifying soybean genotype in soybean breeding;
x15) the use of the product for detecting soybean genotype;
x16) the application of the product in detecting the nutritional value of soybean seeds;
x17) application of the product in detecting the content of the methionine in the soybean seeds;
x18) the use of the product for detecting the content of 11S globulin and/or 7S globulin in soybean seeds;
x19) the use of the product in detecting the ratio of 11S globulin to 7S globulin contents in soybean seeds;
x20) the use of the product in soybean breeding.
The present invention also provides a method of soybean breeding, the method comprising: and (3) identifying the soybean genotype according to the method for identifying the soybean genotype, and selecting the soybean with the AA or GA genotype as a parent to breed.
In the present invention, the sulfur-containing amino acid may be methionine and/or cysteine.
In the present invention, the soybean may be selected from F2 in hybrid progeny of chinese soybean 608 and other soybeans and the pedigree of its later generation.
The additional soybean may be denatology No. 1.
In the present invention, the 7S globulin content may be the content of three subunits, α' and β. The 11S globulin content may be A3Content of three subunits, acid and Basic. The 11S/7S ratio can be A3The ratio of the content of the three subunits, namely, the Acidic subunit and the Basic subunit to the content of the three subunits, namely, the alpha subunit, the alpha' subunit and the beta subunit.
Experiments prove that in the soybean seed protein, the 11S/7S ratios of GG genotypes are all 1.76, the 11S/7S ratios of GA genotypes are averagely 1.91, the 11S/7S ratios of AA genotypes are averagely 2.46, the 11S/7S ratios of the GG genotypes are both obviously lower than those of the GA genotypes and the AA genotypes, and the 11S/7S ratios of the GA genotypes are obviously lower than those of the AA genotypes. The three genotypes of the soybean are related to the 11S/7S ratio in the seed protein, and the 11S/7S ratio in the seed protein can be detected by detecting the soybean molecular marker and the corresponding genotypes. The previous research shows that soybean 7S globulin and 11S globulin are in a significant negative correlation relationship, when the content of 7S globulin is reduced, the content of 11S globulin is increased, and further the content of sulfur-containing amino acid is increased, so that the soybean molecular marker provided by the invention can be used for effectively identifying materials with low 7S globulin content and high sulfur-containing amino acid content. In breeding, the detection of the 11S/7S ratio can be helpful for screening materials with high content of sulfur-containing amino acid so as to improve the nutritional value of the soybeans.
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FIG. 1 shows the SDS-PAGE results of the total protein of seeds of Zhongpin 661 and Zhonghuang 608.
FIG. 2 is F2And (5) generating results of partial plants.
FIG. 3 is F2And (5) generating results of partial plants.
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 experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA.
Soybean Medium yellow 608 in the following examples is given under the accession number ZDD27366 from the national crop germplasm pool (website: http:// www.cgris.net).
The soybean denmark No. 1 in the following examples is a variety approved by the country in 2009, with the variety approval number: bean 2009001.
Example 1 detection of the content of Sulfur-containing amino acids in Soybean seeds
In the embodiment, the sulfur-containing amino acid content of soybean seeds is detected by using a soybean molecular marker, wherein the soybean molecular marker is a DNA molecule obtained by amplifying A1 by using a primer with a soybean genome DNA as a template; a1 is composed of two single-stranded DNAs named A1-F and A1-R, A1-F is the single-stranded DNA shown in the 1 st-20 th position of the sequence 1, A1-R is the single-stranded DNA reverse-complementary to the 1578 th-1598 th position of the sequence 1. The polymorphism of the soybean molecular marker is that a sequence corresponding to a sequence 1 in a soybean genome is a sequence 1 or a sequence 2, the 346 th position of the sequence 1 is G, the 346 th position of the sequence 2 is A, and the sequences 1 and 2 are identical except for the 346 th position.
Accordingly, soybeans are classified into different genotypes, for example, the soybean genome is divided into two chromosomes of g1) and the soybean is divided into GG genotypes; for example, the two chromosomes in the soybean genome are g2) as shown below, and the soybean is of an AA genotype; for example, in the soybean genome, one of two chromosomes is the chromosome of g1) and the other is the chromosome of g2), and soybean is GA genotype;
g1) the nucleotide corresponding to 346 th site of a sequence 1 in a sequence table is G;
g2) the nucleotide corresponding to the 346 th site of the sequence 1 in the sequence table is A.
1. And (3) soybeans to be detected:
soybean intermediate 661; soybean medium yellow 608; soybean denudation No. 1; soybean medium yellow 608 and soybean denudation No. 1F 2 (soybean medium yellow 608 and soybean denudation No. 1 are crossed to obtain F1, and F1 is selfed to obtain F2) were randomly selected from 200 individuals.
2. Detection of genotype
Extracting the genome DNA of each soybean seed to be detected, and detecting the genotype by the following method:
2.1 detection of the genotype of the Soybean to be detected by primer pair A1
And carrying out PCR amplification on the genome DNA of the soybean to be detected by using the primer pair A1, and detecting the genotype of the soybean to be detected.
The PCR amplification system is as follows: DNA template, 4. mu.l (30 ng/. mu.l); KOD-Plus-Neo, 0.4. mu.l; 10 × Buffer, 3.0 μ l; 2.0mM dNTPs, 3.0. mu.l; 25mM MgSO4，1.8μl；10μM A1-F，1.0μl；10μM A1-R，1.0μl；ddH2O, 15.8 μ l; the total volume was 30. mu.l. Wherein KOD-Plus-Neo and 10 xBuffer are products of Beijing Bailingke biotechnology Limited liability company respectively.
PCR reaction procedure: firstly, 94 ℃ for 5 min; then, carrying out 36 cycles of 94 ℃ 30S, 55 ℃ 30S and 72 ℃ 30S; finally, extension is carried out for 10min at 72 ℃, and heat preservation is carried out at 4 ℃.
After the PCR is finished, sequencing the PCR product of each soybean to be detected, and determining the genotype of each soybean to be detected, wherein the results are shown in Table 2.
2.2 detecting the genotype of the soybean to be detected by using a primer pair A2 and a restriction enzyme Taq I
A primer pair A2 is designed according to soybean molecular markers, and A2 consists of two single-stranded DNAs with the names of A2-F (with the sequence 5'-TCTCATTTGGCATTGCGTATCG-3' and the sequence 3 in a sequence table) and A2-R (with the sequence 5'-TTGACCTTCTTCGCATTC-3' and the sequence 4 in the sequence table). When GG genotype soybean was amplified by primer pair A2, the obtained PCR product did not contain the recognition sequence of restriction enzyme Taq I (T ↓; T ↓) in the sequenceCGA, the underlined position is a mutant base, and ↓indicatesthe enzyme digestion position), and the enzyme digestion cannot be cut by Taq I; on the other hand, T (variant 2) forms a recognition sequence for Taq I, which is a restriction enzyme, and thus can be cleaved by Taq I.
According to the principle, firstly, a primer pair A1 is used for carrying out PCR amplification on genome DNA of the soybean to be detected, then restriction enzyme Taq I is used for carrying out enzyme digestion on a PCR amplification product, and the enzyme digestion product is detected, wherein the enzyme digestion product contains a strip with the size of 158bp, the soybean to be detected is GG genotype soybean, the enzyme digestion product contains strips with the sizes of 158bp, 20 bp and 138bp, the soybean to be detected is GA genotype soybean, the enzyme digestion product contains strips with the sizes of 20 bp and 138bp, and the soybean to be detected is AA genotype soybean.
And (3) PCR amplification system: DNA template 4. mu.l (20 ng/. mu.l); easy Taq DNA Polymerase 0.3. mu.l; 10 × Easy Taq Buffer 3 μ l; 2.5mM dNTPs 2.5. mu.l; A2-F3.0. mu.l (2. mu.M); A2-R3.0. mu.l (2. mu.M); ddH2O to 30. mu.l. Wherein, Easy Taq enzyme and 10 × Easy Taq Buffer are all products of Beijing Quanjin Biotechnology Limited. .
PCR reaction procedure: firstly, 94 ℃ for 5 min; then, carrying out 36 cycles of 94 ℃ 30S, 55 ℃ 30S and 72 ℃ 30S; finally, extension is carried out for 10min at 72 ℃, and heat preservation is carried out at 4 ℃.
Enzyme digestion system: 1 mu g of PCR amplification product; restriction enzyme Taq I1. mu.l; 10 XNEBuffer 5. mu.l; ddH2O is added until the total volume is 50 mu l. Compared with the amount of DNA to be enzyme-cut, the sufficient amount of Taq I enzyme in an enzyme cutting system is ensured. Enzyme cutting conditions are as follows: bathing at 65 deg.C for 1 h; after the reaction is finished, the reaction is finished by carrying out warm bath at 80 ℃ for 20 min.
And (3) carrying out 2% agarose gel electrophoresis detection on the enzyme digestion product again, and then determining the genotype of the soybean to be detected according to the electrophoresis result, wherein the result shows that the genotype of the soybean to be detected determined by the method is completely the same as the result in the step 2.1.
3. Detection of soybean seed 7S globulin content
Extracting total protein of each soybean seed to be detected, performing SDS-PAGE (figure 1-3) on the obtained total protein, and quantitatively analyzing target bands in the SDS-PAGE result by using Quantity One software to determine alpha, alpha', beta and 11S globulin subunits A in the seeds3The 11S/7S ratio (table 2) was calculated based on the contents of acid and Basic (table 1), and the 11S/7S ratio was (content of α + content of β)/(a)3Content + acid content + Basic content). Data are in OD mm using Band analysis.
TABLE 2, results of detection of three protein subunits of 11S globulin and three protein subunits of 7S globulin (OD. about. mm)
As can be seen from Table 2, in the soybean seed protein, the GG genotype 11S/7S ratio was 1.76 on average, the GA genotype soybean 11S/7S ratio was 1.91 on average, and the AA genotype soybean 11S/7S ratio was 2.46 on average. The results show that the 11S/7S ratio in the GG genotype soybean seed protein is obviously lower than that of the GA genotype and the AA genotype, and the 11S/7S ratio in the GA genotype soybean seed protein is obviously lower than that of the AA genotype. The three genotypes of the soybean are related to the 11S/7S ratio in the seed protein, and the 11S/7S ratio in the seed protein can be detected by detecting the soybean molecular marker and the corresponding genotypes. The previous research shows that soybean 7S globulin and 11S globulin are in a significant negative correlation relationship, when the content of 7S globulin is reduced, the content of 11S globulin is increased, and further the content of sulfur-containing amino acid is increased, so that the soybean molecular marker provided by the invention can be used for effectively identifying materials with low 7S globulin content and high sulfur-containing amino acid content. In breeding, the detection of the 11S/7S ratio can be helpful for screening materials with high content of sulfur-containing amino acid so as to improve the nutritional value of the soybeans.
<110> institute of crop science of Chinese academy of agricultural sciences
<120> soybean molecular marker and application thereof in detecting nutritional value of soybean seeds
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 1598
<212> DNA
<213> Soybean
<400> 1
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ctgcatgcca ccccgtggcc aaatgtccat gcatgttaac aagacctatg actataaata 180
tctgcaatct cggcccaggt tttcatcatc aagaaccagt tcaatatcct agtacaccgt 240
attaaagaat ttaagatata ctatgatgag agcgcggttc ccattactgt tgctgggagt 300
tgttttcctg gcatcagttt ctgtctcatt tggcattgcg tattgggaaa agcagaaccc 360
cagtcacaac aagtgcctcc gaagttgcaa tagcgagaaa gactcctaca ggaaccaagc 420
atgccacgct cgttgcaacc tccttaaggt ggaggaagaa gaagaatgcg aagaaggtca 480
aattccacga ccacgaccac aacacccgga gagggaacgt cagcaacacg gtgagaagga 540
ggaagacgaa ggtgagcagc cacgtccatt cccattccca cgcccacgcc aacctcatca 600
agaggaagag cacgagcaga aggaggaaca cgaatggcat cgcaaggagg aaaaacacgg 660
aggaaaggga agtgaagagg aacaagatga acgtgaacac ccacgcccac accaacctca 720
tcaaaaggaa gaggaaaagc acgaatggca acacaagcag gaaaagcacc aaggaaagga 780
aagtgaagaa gaagaagaag accaagacga ggatgaggag caagacaaag agagccaaga 840
aagtgaaggt tctgagtctc aaagagaacc acgaagacat aagaataaga acccttttca 900
cttcaactct aaaaggttcc aaactctctt caaaaaccaa tatggccacg ttcgcgtcct 960
ccagaggttc aacaaacgct cccaacagct tcagaatctc cgagactacc gcattttgga 1020
gttcaactcc aaacccaaca cccttcttct cccccaccat gctgacgctg attacctcat 1080
cgttatcctt aacggttagt attcctttcc tctcaaaata aaataaatgt cattatttta 1140
tgcggattaa gaaaaattat taaataaata aaaacaaaac caacaatttt ataaaaacta 1200
atcttactat gacacttatt taattaatta ataaacatca tcacatgctt ttacttctca 1260
taaaattatg ttattgaatt tgttttgtta attacaggga ctgccattct taccttggtg 1320
aacaacgacg accgagactc ttacaacctt caatctggcg atgccctaag agtccctgca 1380
ggaaccacat actatgtggt taaccctgac aacgacgaga atctcagaat gataacactc 1440
gccatacccg ttaacaaacc cggtagattt gaggtactac ttattcttta taactaatta 1500
attaattatt attctaattt gtttccaaat cttaagatca atctttttct tttctctgca 1560
gagtttcttc ctatctagca ctcaagctca acagtcct 1598
<210> 2
<211> 1598
<212> DNA
<213> Soybean
<400> 2
agcccaaaac attcaccaac tcaacccatc atgagcccac acatttgttg tttctaaccc 60
aacctcaaac tcgtattctc ttccgccacc tcatttttgt ttatttcaac acccgtcaaa 120
ctgcatgcca ccccgtggcc aaatgtccat gcatgttaac aagacctatg actataaata 180
tctgcaatct cggcccaggt tttcatcatc aagaaccagt tcaatatcct agtacaccgt 240
attaaagaat ttaagatata ctatgatgag agcgcggttc ccattactgt tgctgggagt 300
tgttttcctg gcatcagttt ctgtctcatt tggcattgcg tattgagaaa agcagaaccc 360
cagtcacaac aagtgcctcc gaagttgcaa tagcgagaaa gactcctaca ggaaccaagc 420
atgccacgct cgttgcaacc tccttaaggt ggaggaagaa gaagaatgcg aagaaggtca 480
aattccacga ccacgaccac aacacccgga gagggaacgt cagcaacacg gtgagaagga 540
ggaagacgaa ggtgagcagc cacgtccatt cccattccca cgcccacgcc aacctcatca 600
agaggaagag cacgagcaga aggaggaaca cgaatggcat cgcaaggagg aaaaacacgg 660
aggaaaggga agtgaagagg aacaagatga acgtgaacac ccacgcccac accaacctca 720
tcaaaaggaa gaggaaaagc acgaatggca acacaagcag gaaaagcacc aaggaaagga 780
aagtgaagaa gaagaagaag accaagacga ggatgaggag caagacaaag agagccaaga 840
aagtgaaggt tctgagtctc aaagagaacc acgaagacat aagaataaga acccttttca 900
cttcaactct aaaaggttcc aaactctctt caaaaaccaa tatggccacg ttcgcgtcct 960
ccagaggttc aacaaacgct cccaacagct tcagaatctc cgagactacc gcattttgga 1020
gttcaactcc aaacccaaca cccttcttct cccccaccat gctgacgctg attacctcat 1080
cgttatcctt aacggttagt attcctttcc tctcaaaata aaataaatgt cattatttta 1140
tgcggattaa gaaaaattat taaataaata aaaacaaaac caacaatttt ataaaaacta 1200
atcttactat gacacttatt taattaatta ataaacatca tcacatgctt ttacttctca 1260
taaaattatg ttattgaatt tgttttgtta attacaggga ctgccattct taccttggtg 1320
aacaacgacg accgagactc ttacaacctt caatctggcg atgccctaag agtccctgca 1380
ggaaccacat actatgtggt taaccctgac aacgacgaga atctcagaat gataacactc 1440
gccatacccg ttaacaaacc cggtagattt gaggtactac ttattcttta taactaatta 1500
attaattatt attctaattt gtttccaaat cttaagatca atctttttct tttctctgca 1560
gagtttcttc ctatctagca ctcaagctca acagtcct 1598
<210> 3
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 3
tctcatttgg cattgcgtat cg 22
<210> 4
<211> 18
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 4
ttgaccttct tcgcattc 18
Claims (14)
1. Soybean molecular marker, using soybean genome DNA as template, adopting primer pair A1 to make amplification to obtain DNA molecule whose sequence is sequence 1 or sequence 2; the A1 consists of two single-stranded DNAs with the names of A1-F and A1-R respectively, the A1-F is the single-stranded DNA which is specifically combined with the position 346 upstream of the sequence 1 in the soybean genome, and the A1-R is the single-stranded DNA which is specifically combined with the position 346 downstream of the sequence 1 in the soybean genome.
2. The soybean molecular marker of claim 1, wherein: the A1-F is single-stranded DNA shown in the 1 st-20 th position of the sequence 1, and the A1-R is single-stranded DNA reverse-complementary to the 1578 th-1598 th position of the sequence 1.
3. The soybean molecular marker of claim 1 or 2, wherein: the soybean is selected from F2 in hybrid progeny of Zhonghuang 608 and other soybeans and the family of its later generations.
4. A method for identifying genotypes of soybean, wherein the genotypes comprise a GG genotype, a GA genotype and an AA genotype, the method comprising the following i or ii or iii or iv:
i, detecting 346 th nucleotide corresponding to a sequence 1 in a sequence table in a soybean genome to be detected, wherein the soybean to be detected is a GG genotype if two chromosomes in the soybean genome to be detected are g 1); if the two chromosomes in the genome of the soybean to be detected are the chromosomes of g2), the soybean to be detected is an AA genotype; if one of the two chromosomes in the genome of the soybean to be detected is the chromosome of g1) below and the other chromosome is the chromosome of g2) below, the soybean to be detected is GA genotype;
g1) the nucleotide corresponding to 346 th site of a sequence 1 in a sequence table is G;
g2) the nucleotide corresponding to 346 th site of a sequence 1 in a sequence table is A;
II, the following K1) and K2):
K1) taking the soybean genome DNA to be detected as a template, and carrying out PCR amplification on the soybean genome DNA by adopting a primer pair A1 to obtain a PCR product; the primer pair A1 consists of two single-stranded DNAs with the names of A1-F and A1-R respectively, wherein A1-F is the single-stranded DNA shown in the 1 st-20 th position of the sequence 1, and A1-R is the single-stranded DNA which is reversely complementary with the 1578 th-1598 th position of the sequence 1;
K2) detecting the sequence of the PCR product obtained in the step K1), and determining the soybean genotype according to the PCR product:
the genotype of the PCR product corresponding to the soybean to be detected with G at the 346 th site of the sequence 1 is GG genotype; the genotype of the PCR product corresponding to the soybean to be detected with G and A at the 346 th site of the sequence 1 is GA genotype; the genotype of the PCR product corresponding to the soybean to be detected, of which the 346 th site of the sequence 1 is A, is an AA genotype;
iii, L1) and L2) as follows:
l1) taking the DNA of the soybean genome to be detected as a template, and carrying out PCR amplification by adopting a primer pair named A2 to obtain a PCR product; the A2 consists of two single-stranded DNAs with the names of A2-F and A2-R respectively, and the sequences of the A2-F and the A2-R are the sequences 3 and 4 in a sequence table respectively;
l2), determining the soybean genotype from the PCR product: the genotype of the PCR product corresponding to the soybean to be detected with G at the 346 th site of the sequence 1 is GG genotype; the genotype of the PCR product corresponding to the soybean to be detected with G and A at the 346 th site of the sequence 1 is GA genotype; the genotype of the PCR product corresponding to the soybean to be detected, of which the 346 th site of the sequence 1 is A, is an AA genotype;
iv, M1) and M2) as follows:
m1) taking the soybean genome DNA to be detected as a template, carrying out PCR amplification by adopting the A2 to obtain a PCR product, and carrying out enzyme digestion on the PCR product by utilizing a restriction enzyme Taq I to obtain an enzyme digestion product;
m2), detecting the size of the enzyme cutting product, and determining the soybean genotype according to the size of the enzyme cutting product: if the enzyme digestion product contains a strip with the size of 158bp, the soybean to be detected is GG genotype soybean, if the enzyme digestion product contains strips with the sizes of 158bp, 20 bp and 138bp, respectively, the soybean to be detected is GA genotype soybean, if the enzyme digestion product contains strips with the sizes of 20 bp and 138bp, the soybean to be detected is AA genotype soybean.
5. The method of claim 4, further comprising: the soybean is selected from F2 in hybrid progeny of Zhonghuang 608 and other soybeans and the family of its later generations.
6. A method for determining the nutritional value of a soybean seed comprising: detecting the genotype of a soybean to be tested by the method of claim 4, wherein the GG genotype soybean seed has a nutritional value lower than or is less than that of a candidate for GA genotype soybean; the nutritive value of GG genotype soybean seeds is lower than that of AA genotype soybean or candidate soybean seeds are lower than that of AA genotype soybean; the nutritional value of GA genotype soybean seeds is lower or the candidate is lower than that of AA genotype soybean.
7. The method of claim 6, wherein: the nutritional value of the soybean seeds is reflected on a1), a2), a3) or a 4):
a1) the content of the sulfur-containing amino acid in the soybean seeds;
a1) the ratio of 11S globulin to 7S globulin content in the soybean seed protein;
a2) 11S globulin content in soybean seed protein;
a3) the 7S globulin content of the soybean seed protein.
8. The method according to claim 6 or 7, wherein: the soybean is selected from F2 in hybrid progeny of Zhonghuang 608 and other soybeans and the family of its later generations.
9. Any of the following applications:
x1) use of the soybean molecular marker of claim 1 or 2 for detecting the nutritional value of soybean seeds;
x2) use of the soybean molecular marker of claim 1 or 2 for detecting the content of the methionine in soybean seeds;
x3) use of the soybean molecular marker of claim 1 or 2 for detecting 11S globulin and/or 7S globulin content in soybean seeds;
x4) use of the soybean molecular marker of claim 1 or 2 for detecting the ratio of 11S globulin to 7S globulin contents in soybean seeds;
x5) use of the soybean molecular marker of claim 1 or 2 in soybean breeding.
10. Use according to claim 9, characterized in that: the soybean is selected from F2 in hybrid progeny of Zhonghuang 608 and other soybeans and the family of its later generations.
11. Any of the following applications:
x6) the use of the method of claim 4 for determining the nutritional value of soybean seeds;
x7) the use of the method of claim 4 for the preparation of a product for testing the nutritional value of soybean seeds;
x8) the use of the method of claim 4 for detecting the content of methionine in soybean seeds;
x9) application of the method in claim 4 in preparing products for detecting the content of the methionine in the soybean seeds;
x10) use of the method of claim 4 for detecting 11S globulin and/or 7S globulin content in soybean seeds;
x11) use of the method of claim 4 for the preparation of a product for detecting 11S globulin and/or 7S globulin content in soybean seeds;
x12) use of the method of claim 4 for determining the ratio of 11S globulin to 7S globulin contents in soybean seeds;
x13) use of the method of claim 4 for the preparation of a product for determining the ratio of 11S globulin to 7S globulin in soybean seeds;
x14) use of the method of claim 4 in soybean breeding.
12. Use according to claim 11, characterized in that: the soybean is selected from F2 in hybrid progeny of Zhonghuang 608 and other soybeans and the family of its later generations.
13. A method of soybean breeding comprising: the method of claim 4, wherein the soybean is genotyped, and soybean of AA or GA genotype is selected as a parent for breeding.
14. The method of claim 13, further comprising: the soybean is selected from F2 in hybrid progeny of Zhonghuang 608 and other soybeans and the family of its later generations.
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| CN105925722A (en) * | 2016-07-11 | 2016-09-07 | 东北农业大学 | Acquisition method of soybean-protein-content-related QTLs (quantitative trait loci) and molecular markers, molecular markers and application thereof |
| CN108060260A (en) * | 2018-01-29 | 2018-05-22 | 吉林省农业科学院 | SNP marker relevant with soya seeds methionine content, section, primer and application |
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| CN105925722A (en) * | 2016-07-11 | 2016-09-07 | 东北农业大学 | Acquisition method of soybean-protein-content-related QTLs (quantitative trait loci) and molecular markers, molecular markers and application thereof |
| CN108060260A (en) * | 2018-01-29 | 2018-05-22 | 吉林省农业科学院 | SNP marker relevant with soya seeds methionine content, section, primer and application |
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