CN116622888B - KASP (KASP-related protein) mark related to soybean glutamic acid and application thereof - Google Patents
KASP (KASP-related protein) mark related to soybean glutamic acid and application thereof Download PDFInfo
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- 235000010469 Glycine max Nutrition 0.000 title claims abstract description 134
- 244000068988 Glycine max Species 0.000 title claims abstract description 133
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 title claims abstract description 55
- 235000013922 glutamic acid Nutrition 0.000 title claims abstract description 55
- 239000004220 glutamic acid Substances 0.000 title claims abstract description 55
- 108090000623 proteins and genes Proteins 0.000 title description 8
- 235000018102 proteins Nutrition 0.000 title description 5
- 102000004169 proteins and genes Human genes 0.000 title description 5
- 239000002773 nucleotide Substances 0.000 claims abstract description 26
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 26
- 238000000034 method Methods 0.000 claims abstract description 25
- 238000009395 breeding Methods 0.000 claims abstract description 19
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- 238000003753 real-time PCR Methods 0.000 claims description 10
- 230000035772 mutation Effects 0.000 claims description 9
- 239000003153 chemical reaction reagent Substances 0.000 claims description 8
- 238000004925 denaturation Methods 0.000 claims description 4
- 230000036425 denaturation Effects 0.000 claims description 4
- 238000004153 renaturation Methods 0.000 claims description 4
- 238000003556 assay Methods 0.000 claims description 3
- 238000012257 pre-denaturation Methods 0.000 claims description 2
- 235000001014 amino acid Nutrition 0.000 abstract description 28
- 150000001413 amino acids Chemical class 0.000 abstract description 28
- 238000003205 genotyping method Methods 0.000 abstract description 15
- 230000000694 effects Effects 0.000 abstract description 8
- 230000008569 process Effects 0.000 abstract description 6
- 230000002349 favourable effect Effects 0.000 abstract description 2
- 238000012214 genetic breeding Methods 0.000 abstract description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 56
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 44
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 31
- 239000004472 Lysine Substances 0.000 description 31
- 239000004471 Glycine Substances 0.000 description 28
- 210000000349 chromosome Anatomy 0.000 description 25
- 239000004475 Arginine Substances 0.000 description 18
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 18
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 18
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 10
- 238000004458 analytical method Methods 0.000 description 8
- 230000007062 hydrolysis Effects 0.000 description 7
- 238000006460 hydrolysis reaction Methods 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 108010073771 Soybean Proteins Proteins 0.000 description 6
- 230000000875 corresponding effect Effects 0.000 description 6
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 5
- 238000012098 association analyses Methods 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 102000039446 nucleic acids Human genes 0.000 description 5
- 108020004707 nucleic acids Proteins 0.000 description 5
- 150000007523 nucleic acids Chemical class 0.000 description 5
- 235000019710 soybean protein Nutrition 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 239000003147 molecular marker Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 238000010219 correlation analysis Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000007844 allele-specific PCR Methods 0.000 description 2
- 230000008045 co-localization Effects 0.000 description 2
- 239000003797 essential amino acid Substances 0.000 description 2
- 235000020776 essential amino acid Nutrition 0.000 description 2
- 102000054766 genetic haplotypes Human genes 0.000 description 2
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- BZTDTCNHAFUJOG-UHFFFAOYSA-N 6-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C11OC(=O)C2=CC=C(C(=O)O)C=C21 BZTDTCNHAFUJOG-UHFFFAOYSA-N 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108010064851 Plant Proteins Proteins 0.000 description 1
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- 239000003517 fume Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000007614 genetic variation Effects 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 238000003976 plant breeding Methods 0.000 description 1
- 235000021118 plant-derived protein Nutrition 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 235000021075 protein intake Nutrition 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 229940001941 soy protein Drugs 0.000 description 1
- 239000010959 steel Substances 0.000 description 1
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Abstract
The invention discloses a KASP (KASP sequence related to soybean glutamic acid) marker and application thereof, belonging to the field of molecular genetic breeding. The KASP is labeled as one or more of s07_6898548 and s12_ 11366121; the nucleotide sequence of the S07_6898548 is shown in SEQ ID NO:3, the nucleotide sequence of the S12_11366121 is shown in SEQ ID NO: shown at 5. The invention develops KASP markers corresponding to the hydrolyzed glutamic acid and the free glutamic acid of the soybeans and performs genotyping, has obvious genotyping effect of samples, can sensitively, efficiently and inexpensively predict the glutamic acid content of the soybeans, is favorable for screening and cultivating soybean varieties with high amino acid content, assists functional molecular breeding of the soybeans, and further shortens the breeding process.
Description
Technical Field
The invention relates to the field of molecular genetic breeding, in particular to a KASP marker related to soybean glutamic acid and application thereof.
Background
The soybean protein content is about 40%, which is one of important sources of human plant protein, and the protein provided accounts for 68% of the total protein consumption worldwide, contains eight essential amino acids which cannot be synthesized by human body, so that the soybean protein has high nutritional value. With the increase of people's diet level, the consumption demand of soybean protein is continuously increasing, and the contradiction between soybean supply and demand is more prominent. Therefore, the research on the rapid and effective soybean protein trait molecular breeding technology has very important production significance for the genetic improvement of the molecular auxiliary soybean protein trait.
In the composition of soy protein, the contents of glutamic acid, lysine, arginine, glycine, and the like are relatively low. The effect of increasing these amino acids by biotechnological means (e.g. transformation) is limited (Altenbach et al, 1987; kortt et al, 1991). Thus, plant breeding methods are being utilized to increase the content of these amino acids in soybeans (Warrington, 2011). Because of the above problems, the soybean breeding community has been targeting the cultivation of soybean varieties with high concentrations of essential amino acids for some time.
Single nucleotide polymorphisms (Single nucleotide polymorphism, SNPs) are widely present at the plant genome level and can be detected by high-throughput techniques, so that these markers are widely used in genetic variation analysis, gene linkage map construction, association mapping, quantitative trait QTL localization, and the like. Competitive allele-specific PCR (Kompetitive Allele Specific PCR, KASP) allows for accurate bi-allele determination of SNPs in a wide range of genomic DNA samples, even some complex genomic DNA samples. The KASP locus specificity is amplified, the mark conversion rate is high, specific mutation loci can be rapidly, accurately and sensitively obtained, the breeding period can be greatly shortened, and the breeding efficiency can be improved. Accordingly, the present invention provides a KASP marker associated with soybean glutamate to meet soybean breeding needs.
Disclosure of Invention
The invention aims to provide a KASP mark related to soybean glutamic acid and application thereof, so as to solve the problems in the prior art, and the KASP mark corresponding to soybean hydrolyzed glutamic acid and free glutamic acid, which is developed by the invention, can sensitively, efficiently and inexpensively predict the soybean glutamic acid content, is beneficial to screening and cultivating soybean varieties with high amino acid content, assists functional molecular breeding of soybean, and further shortens the breeding process.
In order to achieve the above object, the present invention provides the following solutions:
the present invention provides a KASP marker associated with soybean glutamic acid, said KASP marker being one or more of s07_6898548 and s12_ 11366121; the nucleotide sequence of the S07_6898548 is shown in SEQ ID NO:3, the nucleotide sequence of the S12_11366121 is shown in SEQ ID NO: shown at 5.
Further, the 21bp of the S07_6898548 has a G/A base mutation; the 21bp of the S12_11366121 has an A/G base mutation.
The invention also provides a primer group for detecting the KASP mark, and the primer group for detecting the S07_6898548 comprises a nucleotide sequence shown in SEQ ID NO:12, and the nucleotide sequence of the forward primer F1 is shown as SEQ ID NO:13 and the nucleotide sequence of the forward primer F2 is shown as SEQ ID NO:14, a reverse primer R;
the primer group for detecting the S12_11366121 comprises a nucleotide sequence shown in SEQ ID NO:18, and the nucleotide sequence of the forward primer F1 is shown as SEQ ID NO:19 and the nucleotide sequence of the forward primer F2 is shown as SEQ ID NO:20, and a reverse primer R shown in FIG.
The invention also provides a detection reagent or a detection kit marked by KASP, which comprises the primer group.
The invention also provides an application of the KASP mark, the primer group or the detection reagent or the detection kit, which is used in any one of the following applications:
(1) Identifying the glutamic acid content of the soybean;
(2) Screening soybean varieties or strains with high and low glutamic acid content;
(3) Auxiliary breeding of soybean molecular markers;
(4) Improving soybean germplasm resources.
The invention also provides a method for identifying the glutamic acid content of the soybean, which comprises the following steps:
and (3) taking genomic DNA of the soybean sample to be detected as a template, carrying out fluorescent quantitative PCR amplification on the template by using the primer group or the detection reagent or the detection kit, and judging the content of the soybean glutamic acid by using the amplification result.
Further, if the amplification result shows that the base mutation of the S07_6898548 is A, judging that the content of hydrolyzed glutamic acid in the soybean sample to be detected is high; and if the amplification result shows that the base mutation of the S12_11366121 is G, judging that the free glutamic acid content of the soybean sample to be detected is high.
Further, the fluorescent quantitative PCR amplification procedure is as follows: pre-denaturation at 94℃for 15min; denaturation at 94℃for 20s, gradient renaturation/extension at 61-55℃for 1min, 0.6℃decrease per cycle, 10 cycles; denaturation at 94℃for 20s, renaturation/extension at 55℃for 1min,26 cycles.
Further, the fluorescent quantitative PCR amplification system comprises: DNA template 5 u L,2 x KASP Master Mix 5 u L, primer mixture KASP Assay Mix 0.14 u L, total volume 10 u L.
The invention discloses the following technical effects:
the KASP molecular marker has the advantages of low cost, large quantity, obvious genotyping effect and the like, is widely applied to crop breeding, shortens the breeding process, develops the KASP markers corresponding to the hydrolyzed glutamic acid and the free glutamic acid of the soybeans, performs genotyping, and has obvious genotyping effect on samples. The method can sensitively, efficiently and inexpensively predict the glutamic acid content of the soybean, is favorable for screening and cultivating soybean varieties with high amino acid content, assists functional molecular breeding of the soybean, and further shortens the breeding process.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a Manhattan plot and QQ plot of the results of genome-wide association analysis of 4 hydrolyzed amino acid content in soybean kernels; A. b, C, D is the GWAS result of hydrolysis of arginine, glycine, glutamic acid and lysine in sequence;
FIG. 2 is a Manhattan plot and QQ plot of the results of genome-wide association analysis of 4 free amino acid content in soybean kernels; A. b, C, D is the GWAS result of free arginine, glycine, glutamic acid and lysine in turn;
FIG. 3 is a chart of KASP markers genotyping different soybean germplasm SNPs; a is a hydrolyzed arginine genotyping map; b is a hydrolysis glycine genotyping map; c is a hydrolyzed glutamic acid genotyping map; d is a hydrolyzed lysine genotyping map; e is a free glycine, glutamic acid, lysine co-localization genotyping map.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Example 1
The invention provides a SNP locus obviously related to 4 hydrolyzed amino acids of soybean arginine, glycine, glutamic acid and lysine, wherein the average value of the hydrolyzed arginine content of soybean varieties with genotype T is obviously higher than that of soybean varieties with genotype A, and the SNP locus is formed by the steps of (A/T) S16_32280240 (S), S16_36782739 (T/A), S07_6898548 (G/A) and S06_15229665 (A/G), and the free glycine, the glutamic acid and the lysine are co-located S12_11366121 (A/G), so that the corresponding KASP mark is developed; the S16_32280240 locus is shown in SEQ ID NO:1, and the 21 st base of the nucleotide sequence shown in the formula 1;
SEQ ID NO：1：
TGGGTGTTGAGGGGGAGCAAACCAAGCATATGTGCCTCTGTGCACAAGAGGAAAATATTAGCACTCTGACTCTATCTTGTCTAGACGGGGAAACAATTTTCTTGCGATTTTGATTTTGGACTGTCTGTCCTGTTTTTCTCGACATTCTTATTGATGGCAAACCTTTTCATGTGCTAAACTATGACAACTGATTTCTTATAGTCGATTGATTTTCTCGTATTTAGTGAGACAACAAAAATATAGTTTGCATTATGCTGTAGTTATGTGTTAGGTAGTAATACATCTTGTAATAGTATTTTGCTAATATAGTAACGAATACTTACTACCTAACACATAACTACAGCA；
the soybean variety with the S16-36782739 genotype A has higher hydrolysis glycine than the soybean variety with the genotype T; the S16_36782739 locus is shown in SEQ ID NO:2, the 21 st base of the nucleotide sequence shown in the formula 2;
SEQ ID NO：2：
AGTTCTTAGCCCATTTTTTTTTTTTTTATGTTTGTACCAATATGAAAAGAGGTCCTTCAATAGTGCGTATTGTGCCCAGCCTGAGAGCTAAAGATTGGGGCAGTGAATACTATGTGCTGGATGTTTGAATTTGAAATCTTGCAATTGTTTTCTTGGTGACAATATTGATGTATTTTAAAAGCTGATACTTGTTAATTAGAAGGTGTTGAGGTCACTGTATTACGCTTATTATAGTGTTCTCAAGTTTTGAACAATTTGATTGACTTTAAACATAAAACTTGATGGTTATGGAATGAGAAGAAACTCAGAGAGAAACTCAATACAGTGACCTCAACACCTTCT；
the average value of the content of the hydrolyzed glutamic acid of the soybean variety with genotype A at the site S07-6898548 is respectively higher than that of the soybean variety with genotype G; the S07_6898548 locus is shown in SEQ ID NO:3, the 21 st base of the nucleotide sequence shown in the figure;
SEQ ID NO：3：
TTGTATATGGGGTCAACAAAGTCATCATAATTTAAATTGCAAAAAACATATGTTGCAATTGCATGCTAGCAAGGTAGTCGAAGTACTTGAAATTGTCCATAATCACACCACCATTCATTCAAATTGATAGTACATGTTCATTGCTCGTCGACTAAGCCGCGTATTACAATTTCTTGAAACTCAAACTTCCCTGCTTCTCAAACATACATGTGAACACAATGCGAGGTAGCAATGTGTTGATTTTCTTGCATCATGTGACATATACGTCGTGACCAGATTGCCAAATGGAATTATTGAAATAATTATATTGTTCATGGTGAATTGGCAATCTGGTCACGACG；
s06_15229665, the average value of the content of hydrolyzed lysine of the soybean variety with genotype G is higher than that of the soybean variety with genotype A; the s06_15229665 site is set forth in SEQ ID NO:4, the 21 st base of the nucleotide sequence shown in the formula (4);
SEQ ID NO：4：
CTTCATAAGTTTTTTTTAAAATTCGGCTCGGCTTACATAAGAGTCTGACTTGACCCACGAGCCTATTTAAAAGCTTGTTTAAAGACGTCTTTGATTAATTAATTATTTTAAAACCTAATAAAATATTAACTAAAAAAAAGAAACTTATAAAATTTAGTATAAGTAATGTACAAATCCAAAAATAATTGGATAAACAAAATCATATTGAATTCAAGATGTTAAAACACAAAGTATATCAAAAGAAAATAAAAAAAACATAATATTAAAAAATGTATGGATTAAGTCCTCAGCCCCAACCCCAAAGCTTACAAATATATTTTATGTAAGCTTTGGGGTTGGGG；
s12_11366121 locus, the genotype is the soybean variety G, the average value of the content of free glycine, glutamic acid and lysine is higher than that of the soybean variety A; the S12_11366121 locus is shown in SEQ ID NO:5, the 21 st base of the nucleotide sequence shown in the formula (I);
SEQ ID NO：5：
CCTTAACAACAATCCACCTAACAACAGCCTTGGATTTAGGTTGTGGAGTATCATCAAGTCCTTTCAAAGCACTAGTGTTGAATATGAGGAACATTAAAATATTGATGTTGTTGAGTTATGCCAATGAAAATGTATTGTAAACAATACTGACCTGTTAATTATTCCCTTAAGGTAGTTGTCTGGCCTATTATGTAAAGAACAAAACCAGACAACTAGGTTTTCCTTAGGCAGAATGACGACCATTTGCCAGTGTCCGCTGCAGTAGAGACGAATATGGGTTATTGTAGTAGTATAGATTATTTAAATTCAATTATTGTGTTTACACTGGCAAATGGTCGTCA。
note that: the shading of each of the above sequences is marked as a mutant base site.
The molecular marker is specifically obtained according to the following method:
1 materials and methods
1.1 plant Material
The subject group collected, assessed and identified 1084 soybean germplasm. 264 parts of soybean (52 parts of the indigenous seeds and 212 parts of the cultivar) from the whole country were selected, and 19 parts of the wild seeds were collected to constitute 283 parts of the micro-core germplasm resources (Table 1).
Table 1283 numbering and designation of soybean germplasm resources
1.2 major reagents and laboratory apparatus
1.2.1 major reagents
CTAB (Shanghai Jizhi); nucleic acid extract (24:1); 2 x KASP Master mix (LGC, uk); tris (Shanghai Jizhi); EDTA (Shanghai source leaf); beta-mercaptoethanol (Shanghai Jizhi); isopropyl alcohol; PCR specific primers (Peking Optimaceae).
1.2.2 laboratory apparatus
Real-time fluorescent quantitative PCR instrument (nanjing race-fly); plant tissue grinder (nanjing baisi standing grain); centrifuge (Eppendorf, germany); ultra trace nucleic acid protein detector (Beijing Bai Olyman).
1.3 test methods
1.3.1 extraction of soybean DNA
283 parts of fresh young leaves of soybean at the R6 stage are respectively taken, placed into a 2mL centrifuge tube, added with steel balls and preserved by liquid nitrogen. The centrifuge tube is put on a grinder (40 Hz 30 s) for grinding and crushing for standby.
The DNA of soybean leaves is extracted by using a CTAB method. The 100mL configuration system of the 2 XCTAB buffer is shown in Table 2:
table 210ml configuration system of 2 XCTAB buffer
The soybean DNA extraction by using the CTAB method comprises the following steps:
(1) 100mL of a 2 XCTAB buffer was placed in a fume hood and 2% beta-mercaptoethanol was added.
(2) In a soybean sample centrifuge tube after liquid nitrogen freezing and grinding, 800 mu L of 2 XCTAB buffer (2% beta-mercaptoethanol) preheated at 65 ℃ is added, and the mixture is vibrated and mixed uniformly.
(3) The sample was placed in a 65 ℃ water bath for 30min with shaking multiple times.
(4) After the water bath, an equal volume of 800. Mu.L of nucleic acid extract (24:1) was added and shaken for 1min.
(5) The sample was placed in a centrifuge and centrifuged at 10000r/min for 15min, 600. Mu.L of supernatant was taken and placed in a new 1.5mL centrifuge tube.
(6) An equal volume of 600. Mu.L of the nucleic acid extract (24:1) was added to the new centrifuge tube and shaken for 1min.
(7) The sample was placed in a centrifuge at 12000r/min, 400. Mu.L of supernatant was removed and placed in a new 1.5mL centrifuge tube.
(8) 200. Mu.L of 5M NaCl and 400. Mu.L of isopropanol (-20 ℃ C. Precooling) are added into a centrifuge tube, mixed well, and frozen for 30min in a refrigerator at-20 ℃ C.
(9) Taking a precooled sample, putting the precooled sample into a centrifugal machine, centrifuging for 10min at 12000r/min, discarding supernatant, adding 600 mu L of 70% ethanol, and lightly bouncing the precipitate.
(10) Putting into a centrifuge again, centrifuging at 12000r/min for 10min, discarding supernatant, adding 600 μl of 70% ethanol, and bouncing the precipitate.
(11) Putting into a centrifuge, centrifuging at 12000r/min for 10min, uncovering, and putting into a 37 ℃ incubator
(12) After the sample has been completely dried, 50. Mu.L of ddH is added 2 O is dissolved, and the mixture is kept stand for one night at room temperature and stored in a refrigerator at the temperature of minus 20 ℃.
Measuring DNA concentration by nucleic acid protein detector, adding ddH 2 O dilutes the DNA concentration to 50ng/L.
1.3.2 soybean 4 amino acid Whole genome correlation analysis
In this study, SNP markers of the natural population used for GWAS analysis were from the early re-sequencing work of the subject group, and a total of 2,597,425 SNPs were included in the high densityIn the degree physical map (Zhang W, xu W, zhang H, et al, comparative selective signature analysis and high-resolution GWAS reveal a new candidate gene controlling seed weight in soybean [ J)]Theoretical and Applied Genetics,2021,134 (5): 1329-1341.). Whole genome correlation analysis Using a mixed linear model (mLM) and GAPIT package of R software for correlation analysis to reduce false positive SNP sites. Meanwhile, P is selected to be less than or equal to 1/2,597, 425=3.85×10 -7 ，-Log 10 P is more than or equal to 5 as a significant threshold, when SNP is-Log 10 P.gtoreq.5, can be considered a significant association site. From this, SNP sites significantly associated with the 4 hydrolyzed amino acids of soy hydrolyzed arginine, glycine, glutamic acid and lysine, respectively, were identified, s16_32280240 (a/T), s16_36782739 (T/a), s07_6898548 (G/a) and s06_15229665 (a/G), free glycine, glutamic acid, lysine co-located s12_11366121 (a/G).
Development of 1.3.3KASP marker
Primers were designed based on SNP sites S16_32280240 (A/T), S16_36782739 (T/A), S07_6898548 (G/A), S06_15229665 (A/G) which are significantly associated with soybean 4 amino acids, using the Primer-BLAST function of NCBI (https:// www.ncbi.nlm.nih.gov /), three primers were designed for each site, two forward primers F1, F2, one reverse Primer R, wherein F1 and F2 each included a 6-carboxyfluorescein (FAM), hexachloro-6-methylfluorescein (HEX) fluorescent linker sequence (underlined), as shown in Table 3:
TABLE 3 KASP-labeled specific primers
1.3.4KASP labelled amplification system
The KASP labelled 10 μl amplification system comprises: soybean sample DNA template 5. Mu.L (50 ng/. Mu.L), 2 XSKASP Master Mix 5. Mu.L, primer Mix KASP Assay Mix (F1:F2:R=2:2:5) 0.14Mu L, with H 2 O was made up to 10. Mu.L. The KASP-PCR reaction conditions are shown in Table 4, and after the reaction is completed, the product is subjected to fluorescence data reading by a real-time fluorescence quantitative PCR instrument.
TABLE 4KASP-PCR reaction conditions
2. Results and analysis
2.1 Association mapping population resequencing
In the early stage of this group, 283 soybean germplasm was re-sequenced to an average sequencing depth of 12.4×, and 10,210,329 SNP markers were obtained.
2.2 GWAS analysis of soybean 4 amino acids
The phenotype data of the 4 hydrolyzed amino acid contents in the soybean seeds are subjected to genome-wide association analysis and are associated to 343 SNP loci (table 5 and table 7), 21 (-log 10 (P) is more than or equal to 5) SNPs which are obviously related to the arginine contents of the soybean, the SNPs are mainly distributed on chromosome 4, chromosome 5 and chromosome 16, the position of the most obvious SNP is located on chromosome 5 and is 12555523bp, and the maximum value of the log10 (P) is 5.78; the soybean glycine content obviously correlates with 184 SNPs, is mainly distributed on No. 2, 6, 8, 10 and 16 chromosomes, the extremely obvious SNP position is located on No. 6 chromosome and is 12552302bp, and the maximum value of-log 10 (P) value is 12.29; 130 SNPs (single nucleotide polymorphisms) with obvious association of the content of the soybean glutamic acid are mainly distributed on No. 2, 4, 7, 9 and 18 chromosomes, the position of the very obvious SNP is 6735298bp on the No. 7 chromosome, and the maximum value of the-log 10 (P) value is 9.18; the soybean lysine content is obviously related to SNPs, 8 SNPs are mainly distributed on chromosomes 6 and 9, the position of the most obvious SNP is 24573334bp on chromosome 9, and the maximum value of-log 10 (P) is 6.17. The results of the 4 hydrolyzed amino acids GWAS in the soybean kernel are visualized as shown in fig. 1.
The 4 free amino acids in the soybean seeds are correlated to 904 SNP loci (Table 5, table 6), the total number of SNPs with obvious correlation of the content of the free arginine in the soybean is 222 (-log 10 (P) is more than or equal to 5), the SNPs are mainly distributed on chromosome 1, chromosome 4, chromosome 9, chromosome 11 and chromosome 18, the position of the obvious SNP is 11721470bp on chromosome 1, and the maximum value of the log10 (P) is 6.81; the soybean free glycine content is obviously related to the number of SNPs, wherein the number of the SNPs is 491, the SNPs are mainly distributed on the number 1, 8, 11, 12 and 13, the position of the extremely obvious SNP is 20760427bp on the number 12 chromosome, and the maximum value of the-log 10 (P) value is 10.87; the soybean free glutamic acid content is obviously related to the SNPs, wherein the number of the SNPs is 133, the SNPs are mainly distributed on the 10, 11, 12 and 18 chromosomes, the position of the extremely obvious SNP is 53291599bp on the 18 th chromosome, and the maximum value of the-log 10 (P) value is 6.44; there are 58 SNPs with significant correlation of free lysine content in soybean, and the SNPs are mainly distributed on chromosomes 8, 12, 17, 18 and 19, the positions of the very significant SNPs are located on chromosome 8 by 18610062bp, and the maximum value of-log 10 (P) is 6.09. The results of the 4 free amino acids GWAS in soybean kernels are visualized as shown in fig. 2.
As can be seen from Table 8, soybean free glycine, glutamic acid and lysine were co-localized to 3 SNP sites, respectively S12_11366121, S12_11442535, S12_11445996, free arginine and glycine were co-localized to 5 SNP sites, respectively S01_53974257, S01_11721458, S01_11721461, S01_11721464, S01_11721470, wherein the effect of correlating the S12_11366121 sites was remarkable, and KASP markers could be further developed for population genotyping.
TABLE 5 major SNPs with significant correlation of the 4 hydrolyzed amino acid contents of soybean
TABLE 6 major SNPs with significant correlation of the 4 free amino acid contents of soybean kernels
TABLE 7 number of SNP sites associated with soybean 4 amino acid contents
TABLE 8 Co-located SNPs of 4 free amino acids for soybean seeds
2.3 haplotype analysis of different genotypes
As can be seen from Table 9, SNP loci which are obviously related to the arginine, glycine, glutamic acid and lysine of soybean seeds are S16_32280240 (A/T), S16_36782739 (T/A), S07_6898548 (G/A) and S06_15229665 (A/G), and the loci and free glycine, glutamic acid and lysine are co-located S12_11366121 (A/G) and subjected to different genotype haplotype analysis, so that the positions S16_32280240 which are obviously related to the content of the arginine are found, the genotype of the soybean variety is TT with the average value of 56.20mg/G, the genotype of the soybean variety is AA with the average value of 31.42mg/G, and the content of the soybean variety is improved by 78.86; the hydrolysis glycine content is obviously related to site S16_36782739, the average value of the content of AA in the soybean variety is 45.63mg/g, the average value of the content of TT in the soybean variety is 16.11mg/g, and the content is improved by 183.24%; the content of the hydrolyzed glutamic acid is obviously related to site S07_6898548, the average value of the content of AA in the soybean variety is 125.54mg/g, the average value of the content of GG in the soybean variety is 46.89mg/g, and the content is improved by 167.73% on average; the content of the hydrolyzed lysine is obviously related to site S06_15229665, the average value of the content of the genotype GG in the soybean variety is 65.26mg/g, the average value of the content of the genotype AA is 39.43mg/g, and the content is improved by 65.50 percent on average; the average value of the content of the genotype GG in the soybean variety is 0.20mg/g, 0.93mg/g and 0.17mg/g, the average value of the content of the genotype AA is 0.07mg/g, 0.52mg/g and 0.11mg/g, and the average content is increased by 185.71%, 78.84% and 54.55%.
TABLE 9 amino acid content of soybean germplasm of different genotypes
2.3 soybean 4 amino acid KASP marking application
Corresponding KASP markers were developed for SNP sites s16_32280240 (a/T), s16_36782739 (T/a), s07_6898548 (G/a) and s06_15229665 (a/G) and free glycine, glutamic acid, lysine co-localization s12_11366121 (a/G) that are significantly associated with soybean kernel hydrolysis arginine, glycine, glutamic acid, lysine, and the marker primer sequences are shown in table 2. Application procedure of KASP flag: firstly, extracting DNA of soybean genome as a template, adding corresponding primers F1, F2 and R of each locus, performing PCR amplification in a real-time fluorescent quantitative PCR instrument, and finally, reading fluorescent data after the reaction is finished. In this study, 25 parts of soybean germplasm (the quality of the content of each amino acid of which is known in soybean germplasm is shown in tables 10-11) is amplified and genotyped by using a KASP marker in a real-time fluorescence quantitative PCR instrument, and the result is shown in figure 3, and the two genotypes can be clearly distinguished by 4 molecular marker primers.
Table 1025 amino acid content of soybean germplasm and genotyping results
TABLE 11
The soybean hydrolyzed arginine content is obviously related to KASP markers designed by SNP locus S16_32280240 (A/T), two genotypes can be clearly distinguished, blue dots are soybean varieties with high arginine content and T allelic variation loci, and red dots are soybean germplasm with general A allelic variation locus content (A in figure 3); the hydrolysis glycine content is obviously related to KASP markers designed by SNP locus S16_36782739 (T/A), blue dots are soybean varieties with high glycine content and carrying A allelic variation loci, and red dots are soybean germplasm with general T allelic variation locus content (B in figure 3); the hydrolyzed glutamic acid content is obviously related to KASP markers designed by SNP locus S07_6898548 (G/A), blue dots are soybean varieties with high glutamic acid content and carrying A allelic variation loci, and red dots are soybean germplasm with general G allelic variation loci (C in figure 3); the hydrolyzed lysine content is obviously related to KASP markers designed by SNP locus S06_15229665 (A/G), blue dots are soybean varieties with high lysine content and G allelic variation loci, and red dots are soybean germplasm with common A allelic variation locus content (D in figure 3); free glycine, glutamic acid and lysine co-localize SNP sites s12_11366121 (a/G) KASP markers were designed, blue dots are soybean varieties with high free glycine, glutamic acid and lysine content carrying G allelic variation sites, red dots are soybean germplasm carrying a general content of a allelic variation sites (E in fig. 3).
3 analysis of results
The study carries out whole genome association analysis on soybean natural populations, identifies SNP loci which are obviously associated with 4 hydrolyzed amino acids of soybean arginine, glycine, glutamic acid and lysine respectively, wherein the average value of the hydrolyzed arginine content of soybean varieties with genotype T is obviously higher than that of soybean varieties with genotype A; the soybean variety with the S16-36782739 genotype A has higher hydrolysis glycine than the soybean variety with the genotype T; the average value of the content of the hydrolyzed glutamic acid of the soybean variety with genotype A at the site S07-6898548 is respectively higher than that of the soybean variety with genotype G; s06_15229665, the average value of the content of hydrolyzed lysine of the soybean variety with genotype G is higher than that of the soybean variety with genotype A; s12_11366121 locus, the genotype is the soybean variety G with the average content of free glycine, glutamic acid and lysine higher than that of the soybean variety A.
photosensitive (2020) hydrolyzed amino acid content determination and genome-wide association analysis of the middle and south soybean test population showed that the determination result in 2018 was in-LOG 10 P>When 3 is a significant level, the number of significant SNP markers of lysine on chromosome 6 is 20, the most significant site is S6_51390910, the most significant SNP site of lysine in the study is also located on chromosome 6, and due to different significance thresholds, the study is LOG 10 P>Fewer SNP sites are located on chromosome 5, 6. Based on SNP locus on lysine 6 chromosome, can further explore the related candidate gene of soybean lysine content, lay the foundation for the subsequent functional soybean molecular breeding.
The KASP molecular marker has the advantages of low cost, large quantity, obvious genotyping effect and the like, is widely applied to crop breeding, shortens the breeding process, and has obvious genotyping effect on samples after researching and developing KASP markers corresponding to 4 amino acids of soybean arginine, glycine, glutamic acid and lysine. Can sensitively, efficiently and inexpensively predict the content of soybean amino acid and promote the breeding process of soybean molecules.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (8)
1. A KASP marker associated with soybean glutamic acid, wherein the KASP marker is one or more of s07_6898548 and s12_ 11366121; the nucleotide sequence of the S07_6898548 is shown in SEQ ID NO:3, the nucleotide sequence of the S12_11366121 is shown in SEQ ID NO:5 is shown in the figure;
a G/A base mutation is arranged at the 21bp of the S07_ 6898548; the 21bp of the S12_11366121 has an A/G base mutation.
2. A primer set for detecting the KASP marker of claim 1, wherein the primer set for detecting s07_6898548 comprises a nucleotide sequence set forth in SEQ ID NO:12, and the nucleotide sequence of the forward primer F1 is shown as SEQ ID NO:13 and the nucleotide sequence of the forward primer F2 is shown as SEQ ID NO:14, a reverse primer R;
the primer group for detecting the S12_11366121 comprises a nucleotide sequence shown in SEQ ID NO:18, and the nucleotide sequence of the forward primer F1 is shown as SEQ ID NO:19 and the nucleotide sequence of the forward primer F2 is shown as SEQ ID NO:20, and a reverse primer R shown in FIG.
3. A KASP-tagged detection reagent or detection kit according to claim 1, comprising the primer set of claim 2.
4. Use of a KASP marker according to claim 1, a primer set according to claim 2 or a detection reagent or kit according to claim 3, for use in any of the following applications:
(1) Identifying the glutamic acid content of the soybean;
(2) Screening soybean varieties or strains with high and low glutamic acid content;
(3) Auxiliary breeding of soybean molecular markers;
(4) Improving soybean germplasm resources.
5. A method for identifying the glutamic acid content of soybean, which is characterized by comprising the following steps:
taking genomic DNA of a soybean sample to be detected as a template, carrying out fluorescent quantitative PCR amplification on the template by using the primer set according to claim 2 or the detection reagent or the detection kit according to claim 3, and judging the content of the soybean glutamic acid by using the amplification result.
6. The method according to claim 5, wherein if the amplification result shows that the base mutation of s07_6898548 in claim 1 is a, the sample to be tested is judged to have a high content of hydrolyzed glutamic acid; if the amplification result shows that the base mutation of S12_11366121 in the method in claim 1 is G, the free glutamic acid content of the soybean sample to be detected is judged to be high.
7. The method of claim 5, wherein the fluorescent quantitative PCR amplification procedure is: pre-denaturation at 94℃for 15min; denaturation at 94℃for 20s, gradient renaturation/extension at 61-55℃for 1min, 0.6℃decrease per cycle, 10 cycles; denaturation at 94℃for 20s, renaturation/extension at 55℃for 1min,26 cycles.
8. The method of claim 5, wherein the fluorescent quantitative PCR amplification system is: DNA template 5 u L,2 x KASP Master Mix 5 u L, primer mixture KASP Assay Mix 0.14 u L, total volume 10 u L.
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| US10041089B1 (en) * | 2014-10-27 | 2018-08-07 | Syngenta Participations Ag | Resistance alleles in soybean |
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| CN105985958A (en) * | 2015-02-06 | 2016-10-05 | 中国科学院上海生命科学研究院 | Specific promoter of leguminous plant leaf tissues and application of specific promoter |
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