CN116837128A - SNP molecular marker extremely remarkably related to sucrose content of vegetable soybean seeds and application thereof - Google Patents
SNP molecular marker extremely remarkably related to sucrose content of vegetable soybean seeds and application thereof Download PDFInfo
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- CN116837128A CN116837128A CN202310684785.8A CN202310684785A CN116837128A CN 116837128 A CN116837128 A CN 116837128A CN 202310684785 A CN202310684785 A CN 202310684785A CN 116837128 A CN116837128 A CN 116837128A
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- 235000010469 Glycine max Nutrition 0.000 title claims abstract description 57
- 244000068988 Glycine max Species 0.000 title claims abstract description 57
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 title claims abstract description 44
- 229930006000 Sucrose Natural products 0.000 title claims abstract description 44
- 239000005720 sucrose Substances 0.000 title claims abstract description 44
- 235000013311 vegetables Nutrition 0.000 title claims abstract description 39
- 239000003147 molecular marker Substances 0.000 title claims abstract description 25
- 238000009395 breeding Methods 0.000 claims abstract description 9
- 230000001488 breeding effect Effects 0.000 claims abstract description 9
- 239000002773 nucleotide Substances 0.000 claims abstract description 8
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 8
- 238000000034 method Methods 0.000 claims abstract description 7
- 238000012408 PCR amplification Methods 0.000 claims description 4
- 108090000623 proteins and genes Proteins 0.000 abstract description 8
- 238000012098 association analyses Methods 0.000 abstract description 4
- 210000000349 chromosome Anatomy 0.000 abstract description 4
- 238000010219 correlation analysis Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000000105 evaporative light scattering detection Methods 0.000 description 2
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000004807 localization Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 2
- -1 BEH Amide Chemical class 0.000 description 1
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- NNBFNNNWANBMTI-UHFFFAOYSA-M brilliant green Chemical group OS([O-])(=O)=O.C1=CC(N(CC)CC)=CC=C1C(C=1C=CC=CC=1)=C1C=CC(=[N+](CC)CC)C=C1 NNBFNNNWANBMTI-UHFFFAOYSA-M 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000013480 data collection Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000004720 fertilization Effects 0.000 description 1
- 229960000304 folic acid Drugs 0.000 description 1
- 235000019152 folic acid Nutrition 0.000 description 1
- 239000011724 folic acid Substances 0.000 description 1
- 238000012214 genetic breeding Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000003205 genotyping method Methods 0.000 description 1
- GOMNOOKGLZYEJT-UHFFFAOYSA-N isoflavone Chemical compound C=1OC2=CC=CC=C2C(=O)C=1C1=CC=CC=C1 GOMNOOKGLZYEJT-UHFFFAOYSA-N 0.000 description 1
- CJWQYWQDLBZGPD-UHFFFAOYSA-N isoflavone Natural products C1=C(OC)C(OC)=CC(OC)=C1C1=COC2=C(C=CC(C)(C)O3)C3=C(OC)C=C2C1=O CJWQYWQDLBZGPD-UHFFFAOYSA-N 0.000 description 1
- 235000008696 isoflavones Nutrition 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000003044 randomized block design Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000009331 sowing Methods 0.000 description 1
- 235000019605 sweet taste sensations Nutrition 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
The invention discloses an SNP molecular marker extremely obviously related to sucrose content of vegetable soybean seeds and application thereof. In particular to a SNP molecular marker of 1 on soybean chromosome 14 and the sucrose content of vegetable soybean seeds and application thereof. The molecular marker is located in the intron region of gene Glyma.14G192100. The molecular marker is obtained from genome-wide association analysis of soybean local germplasm population, and the nucleotide sequence of the molecular marker is shown as SEQ ID NO 1. The SNP molecular marker can be applied to high-quality breeding of vegetable soybeans, and the breeding process of new varieties is accelerated.
Description
Technical Field
The invention belongs to the technical field of application of crop genetic breeding molecular markers, and particularly relates to a SNP marker related to sucrose content of vegetable soybeans and application thereof.
Background
The vegetable soybeans are also called green soybeans, are the general name of the soybeans harvested when the seeds are full and the color is emerald green, have shorter maturation period and have extremely high economic value. At present, vegetable soybeans are widely cultivated in southeast coastal areas such as Jiangsu, zhejiang, fujian, guangdong and Guangxi, and also partly cultivated in northeast and southwest areas. The vegetable soybean has fresh and sweet taste and rich nutrition, is rich in nutrients such as protein, isoflavone, folic acid, trace elements and the like, and has important edible and health care values. The sucrose content of the soybean milk affects the taste and the nutrition quality of the vegetable soybean, is also an important index for classifying commercial vegetable soybean, and is also a core requirement for variety breeding.
Sucrose content of vegetable soybeans is a typical quantitative trait, has higher genetic power, and plays a role in major genes with larger influence on the trait. For the sucrose content of crops, the conventional mapping mode is generally adopted for gene localization, but the localization interval is larger, and the target gene is difficult to find.
Disclosure of Invention
The invention aims to provide an SNP molecular marker which is extremely obviously related to the sucrose content of vegetable soybean seeds and application thereof.
The invention provides a SNP molecular marker related to sucrose content of vegetable soybean seeds, wherein the molecular marker is that a T/G polymorphism site exists at the 151 st position of a sequence with a nucleotide sequence shown as SEQ ID NO.1, genotype G/G indicates that the sucrose content of the soybean seeds is high, and genotype T/T indicates that the sucrose content of the soybean seeds is low.
According to the invention, the sucrose content of the seed grains in the fresh eating period of the regional germplasm population of the middle soybean is measured, and 82187 SNP markers are utilized to carry out whole genome association analysis, so that the SNP obviously related to the regional germplasm population of the middle soybean is identified on chromosome 14. The SNP marker is positioned on a nucleotide sequence shown in SEQ ID NO.1, and the 151 th base from the 5' end of the sequence is a SNP locus; the SNP is located at the 45682176 (bp) position of the soybean chromosome 14 and falls within the intron region of the Glyma.14G192100 gene.
The invention also provides a primer or a kit for amplifying the SNP molecular marker.
The invention also provides application of the SNP molecular marker or a primer or a kit for amplifying the SNP molecular marker in identifying or assisting in identifying sucrose of vegetable soybean seeds or breeding of vegetable soybean seeds.
A method for identifying sucrose content of vegetable soybean seeds specifically comprises detecting genotype of SNP molecular markers.
The genotype of the SNP molecular marker according to the invention can be detected by the conventional methods in the art, such as PCR amplification.
The invention compares samples of different genotypes at a significant site, with an inter-group mean difference of 2.50mg/g (P < 0.01) at that site. Thus, genotype G/G is a very significant molecular marker with a relatively high sucrose content, typically greater than 17mg/G sucrose content, and genotype T/T is a very significant molecular marker with a relatively low sucrose content, typically less than 15mg/G sucrose content.
The invention discovers SNP molecular markers related to sucrose content of vegetable soybeans by means of whole genome association analysis, and uses the SNP molecular markers as molecular markers adopted in quality breeding of vegetable soybeans so as to accelerate the process of vegetable soybean variety breeding.
Drawings
FIG. 1 is a Manhattan plot of a whole genome correlation analysis of sucrose content of vegetable soybean kernels.
FIG. 2 is a Q-Q plot of a genome-wide correlation analysis of sucrose content of vegetable soybean kernels.
Detailed Description
The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.
Example 1 phenotypic data collection
1.1 planting of germplasm Material
The 133 germplasm used in the invention is planted in the Jiang Ji places of Anhui province academy of agricultural sciences and Nanjing university of agriculture in 2021 and 2022. Both trials were performed with a completely randomized block design, with a total of three replicates. Any germplasm in each repetition is provided with 1 field planting number, and the group, repetition and germplasm names are noted. The row length is set to be 1 meter, the row spacing is 0.5 meter, the plant spacing is 0.2 meter, and 6 plants are planted in each row. Every ten rows are provided with a ground inserting card, and information such as numbers and the like is marked in detail. Other pretreatment before sowing, fertilization and deinsectization measures are equivalent to general field management.
1.2 sample collection
Samples were taken during the period from soybean growth to R6-R7 and 10 pods were taken from each plant at about 9 a.m. from each position, 60 pods were taken per row. After sampling was completed, the samples were immediately snap frozen in liquid nitrogen and then stored in a-40 ℃ refrigerator.
1.3 sucrose content determination
The sucrose content of fresh vegetable soybean seeds was measured using a WATERS ACQUITY UPLC H-Class ultra-high performance liquid chromatograph, UPLC ACQUITY BEH Amide 1.7.7 um x 2.1 x 100mm column and an Evaporative Light Scattering Detector (ELSD), and the parameters of the liquid chromatograph were set as shown in table 1.
Table 1: parameters and conditions of ultra-high performance liquid chromatograph
EXAMPLE 2 Whole genome correlation analysis of sucrose content of vegetable soybeans
The invention uses MLM (PCA+K) model in TASSEL v5.2 software to carry out genome-wide association analysis of sucrose content of vegetable soybean, and sets a threshold value of obvious association between SNP markers and target shapes as-log 10 (P) =3.5, a very significantly related SNP molecular marker was detected at chromosome 14, 45682176 (bp), and combined with linkage disequilibrium attenuation distance 119.07kb, the candidate gene glyma.14g192100 was determined (fig. 1 and 2). The results are detailed in Table 2. The molecule is marked as being in the nucleusThe 151 st position of the nucleotide sequence shown in SEQ ID NO.1 has a T/G polymorphic site, the genotype G/G shows that the sucrose content of the soybean seeds is high, and the genotype T/T shows that the sucrose content of the soybean seeds is low.
Table 2: whole genome correlation analysis of sucrose content of vegetable soybean seeds
Example 3 inter-group comparison of Chr14-45682176
The locus includes 2 genotypes: T/T, G/G. The results of the t-test for differences in the mean phenotypes between the different genotypes are shown in Table 3. The difference between the T/T and G/G is very significant, and the average difference between the groups is 2.5mg/G. Genotype G/G is a molecular marker with high sucrose content, and genotype T/T is a molecular marker with low sucrose content.
Table 3 comparison of mean values between groups
EXAMPLE 4 verification of site availability
To verify the effectiveness of this site, a pair of primers was designed using NCBI website with the sequence comprising SNP sites significantly correlated to sucrose content of the vegetable soybeans obtained by screening in example 2 as a template, the primer sequences were as follows:
forward primer: AAGGGACCTCTGTCATTGGG (SEQ ID NO 2)
Reverse primer: GCCTATAGCCATTGAGCATTCAC (SEQ ID NO 3)
The primers are used for carrying out common PCR amplification on genomic DNA of vegetable soybeans to be screened, and the amplification system is as follows: 2X Rapid Taq Master Mix 12.5.5. Mu.l, 10. Mu.M forward primer 1. Mu.l, 10. Mu.M reverse primer 1. Mu.l, 10. Mu.M DNA template 1. Mu.l ddH 2 O9.5 μl. The reaction procedure of PCR amplification is 95 ℃ for 5min;95 ℃ for 30s, 56 ℃ for 30s,72 ℃ for 30s,34 cycles; and at 72℃for 5min. After obtaining the DNA fragment, sequencing, comparing the sequencing result with the vegetable soybean related gene fragment SEQ ID NO1, and detectingAnd (3) detecting the genotype carried by the SNP locus at the 151 st position of the sequence, so that the sucrose content of the vegetable soybean can be effectively selected.
To verify the practicability of the marker, 20 soybean varieties are randomly selected, and the genotyping and the determination of the sucrose content in the fresh period after sequencing are carried out, and the results are shown in tables 4 and 5, so that the marker has the practicability.
Table 4: sucrose content of 20 different vegetable soybeans
Table 5: group mean comparison of 20 vegetable soybeans
Claims (10)
1. SNP molecular markers related to sucrose content of vegetable soybean seeds, wherein the molecular markers are T/G polymorphic sites exist at the 151 st position of a sequence with a nucleotide sequence shown as SEQ ID NO.1, genotype G/G indicates that the sucrose content of the soybean seeds is high, and genotype T/T indicates that the sucrose content of the soybean seeds is low.
2. Use of the SNP molecular marker of claim 1 for identifying or aiding in the identification of sucrose from vegetable soybean seeds or breeding of vegetable soybean seeds.
3. Amplifying the SNP molecular marker primer of claim 1.
4. The primer according to claim 3, specifically a forward primer having a nucleotide sequence shown as SEQ ID NO. 2 and a reverse primer having a nucleotide sequence shown as SEQ ID NO. 3.
5. Use of the primer of claim 3 or 4 for identifying or assisting in identifying sucrose or seed breeding of vegetable soybean seeds.
6. A kit for amplifying the SNP molecular markers of claim 1.
7. Use of the kit of claim 6 for identifying or aiding in the identification of sucrose from vegetable soybean seeds or breeding of vegetable soybean seeds.
8. A method for identifying sucrose content of vegetable soybean seeds specifically comprises detecting genotype of SNP molecular marker, wherein the molecular marker is T/G polymorphic site at 151 th position of a sequence with a nucleotide sequence shown as SEQ ID NO. 1.
9. The method according to claim 8, wherein the genotype of the SNP molecular marker according to claim 1 is detected by PCR amplification.
10. The method of claim 8, wherein genotype G/G indicates high sucrose content of the soybean kernel and genotype T/T indicates low sucrose content of the soybean kernel.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202310684785.8A CN116837128A (en) | 2023-06-09 | 2023-06-09 | SNP molecular marker extremely remarkably related to sucrose content of vegetable soybean seeds and application thereof |
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| CN202310684785.8A CN116837128A (en) | 2023-06-09 | 2023-06-09 | SNP molecular marker extremely remarkably related to sucrose content of vegetable soybean seeds and application thereof |
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