CN116004900A - Molecular marker AT12 for identifying aluminium-tolerant cause type of tomatoes and application thereof - Google Patents
Molecular marker AT12 for identifying aluminium-tolerant cause type of tomatoes and application thereof Download PDFInfo
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- 235000007688 Lycopersicon esculentum Nutrition 0.000 title claims abstract description 50
- 239000003147 molecular marker Substances 0.000 title claims abstract description 28
- 240000003768 Solanum lycopersicum Species 0.000 title claims description 45
- 229910052782 aluminium Inorganic materials 0.000 claims abstract description 49
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims abstract description 45
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 14
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- 238000000034 method Methods 0.000 claims description 15
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- 241000227653 Lycopersicon Species 0.000 abstract 6
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- 238000001514 detection method Methods 0.000 description 5
- 235000015097 nutrients Nutrition 0.000 description 5
- -1 aluminum ions Chemical class 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
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- 231100000419 toxicity Toxicity 0.000 description 4
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- ODINCKMPIJJUCX-UHFFFAOYSA-N Calcium oxide Chemical compound [Ca]=O ODINCKMPIJJUCX-UHFFFAOYSA-N 0.000 description 2
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- 229920000936 Agarose Polymers 0.000 description 1
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- 238000010353 genetic engineering Methods 0.000 description 1
- 238000003205 genotyping method Methods 0.000 description 1
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Abstract
The invention relates to the technical field of tomato aluminum resistance, in particular to a molecular marker AT12 for identifying an aluminum-resistant cause type of tomatoes and application thereof. The molecular marker takes a tomato SL2.50 genome version as a reference gene, is positioned at a 1338779 site on a chromosome 12, has G/A polymorphism, can effectively screen aluminum-resistant resources in current tomato resources, can quickly and accurately introduce a tomato aluminum-resistant main effect control gene into excellent tomato parents by combining a conventional breeding means, creates new aluminum-resistant germplasm resources, and cultivates new stress-resistant varieties.
Description
Technical Field
The invention relates to the technical field of tomato aluminum resistance, in particular to a molecular marker AT12 for identifying an aluminum-resistant cause type of tomatoes and application thereof.
Background
Aluminum (Al) isOne of the most abundant metallic elements in the soil, approximately 8%, although aluminum is non-toxic in oxides or hydroxides under neutral and alkaline conditions, the solubility of aluminum increases dramatically when the soil pH is below 5.5, and the dissolved aluminum is highly toxic to most plants, with a fraction of trivalent aluminum ions (Al 3+ ) Is dissolved to inhibit the growth of plant roots, thereby affecting the growth and development of crops and finally resulting in the yield reduction of crops. Thus, aluminum toxicity is a major limiting factor for crops in acid soil, and is also the second most abiotic stress next to drought, and with excessive frequent cultivation and excessive use of nitrogen fertilizer, the soil tends to be further acidified, which also makes the problem of aluminum toxicity increasingly serious.
In agriculture, quicklime is usually applied to relieve aluminum toxicity by acid soil, however, the method only can improve surface soil, can not change the pH value of deep soil, and needs a great deal of financial resources and manpower. Whereas plants survive in acidic aluminum-toxic soil, many complex response mechanisms occur during development, mainly controlled by transcriptional regulation in response to Al stress. Therefore, the method for reducing the acidity of the soil and simultaneously excavating the self aluminum resistance potential of the plant to obtain a plant variety with strong aluminum resistance, or utilizing a genetic engineering means to increase the aluminum resistance of the plant is an effective strategy for solving the problems of aluminum toxicity in acid soil and sustainable development of agricultural production.
Tomato is a rich nutrient source and a model plant for the development of fleshy fruits, and is one of important vegetable crops. At present, tomato aluminum-resistant breeding is still in a starting stage, and a conventional breeding method is mainly used, and is time-consuming, poor in accuracy and easy to be interfered by environment. The molecular marker assisted breeding utilizes the characteristic that the molecular marker is closely linked with a target character gene, and the target character is selected by detecting the molecular marker, so that the molecular marker has the advantages of rapidness, accuracy and no interference from environmental conditions, and the molecular marker related to the aluminum tolerance of tomatoes is rarely reported at present.
Disclosure of Invention
Based on the method, the invention analyzes 450 parts of tomato resources, and discovers a general molecular marker for identifying the aluminum tolerance of tomatoes for the first time, wherein the marker takes a SL2.50 genome version of tomatoes as a reference gene, is positioned at a 1338779 site on chromosome 12, and has G/A polymorphism. Wherein G is a genomic sequence and A is a mutant base. The molecular marker can effectively screen aluminum-resistant resources in current tomato resources, simultaneously can be combined with conventional breeding means to rapidly and accurately introduce aluminum-resistant major control genes into excellent tomato parents, creates new stress-resistant germplasm resources, breeds new stress-resistant varieties, and has important application prospects.
The invention also aims AT protecting the primer for amplifying the molecular marker AT12.
As a preferred embodiment, the primer comprises a forward outer primer shown as SEQ ID NO. 3, a reverse outer primer shown as SEQ ID NO. 4, a forward inner primer shown as SEQ ID NO. 5, and a reverse inner primer shown as SEQ ID NO. 6.
The invention also aims at protecting a kit for identifying the aluminium tolerance cause type of tomatoes, which comprises the primer.
The invention also aims at protecting a method for identifying the aluminium-tolerant cause of tomatoes, which comprises the following steps: extracting total DNA of a tomato sample to be detected, amplifying by using the total DNA of the tomato to be detected as a template and a specific primer with a sequence shown as SEQ ID NO. 3-6, and judging according to the length of the amplified fragment;
the bands with the sizes of 199bp and 439bp are aluminum-resistant gene types, the bands with the sizes of 295bp and 439bp are aluminum-intolerant gene types, and the bands with the sizes of 199bp, 295bp and 439bp are heterozygous genotypes.
As a preferred embodiment, the system upon amplification is: 2 XTag mix 9.6. Mu.L, outer primers 0.4. Mu.L each, inner primers 0.6. Mu.L each, DNA template 1. Mu. L, ddH 2 O 7.4μL。
As a preferred embodiment, the procedure at the time of amplification is: pre-denaturation at 94℃for 3min, denaturation at 94℃for 30s, annealing at 55℃for 30s, extension at 72℃for 30s, 36 cycles, extension at 72℃for 5min, cooling to 15 ℃.
The invention also aims to protect the application of the molecular marker and the primer in the identification of the aluminium-tolerant cause of tomatoes.
The invention also aims to protect the application of the molecular marker and the primer in breeding of aluminum-resistant tomato varieties.
The invention also aims to protect the application of the molecular marker and the primer in cultivating aluminum-resistant tomato varieties.
Drawings
FIGS. 1A and 1B are the results of the whole genome correlation analysis in example 1;
FIG. 1C is a 200kb analysis result upstream and downstream of the significant site on chromosome 12 in example 1;
FIG. 1D shows the aluminum content of the aerial parts of plants with three genotypes GG, GA and AA;
FIG. 1E is a comparison of sequencing results for extremely high and extremely low materials;
FIG. 2A is a plant height comparison of four materials of example 3;
FIG. 2B is a root length comparison of four materials of example 3;
FIG. 2C is a phenotypic photograph of the four materials of example 3;
FIG. 3 shows a part of the detection results in example 4.
Detailed Description
The present invention will be described in further detail with reference to specific examples so as to more clearly understand the present invention by those skilled in the art.
The following examples are given for illustration of the invention only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art without creative efforts are within the protection scope of the present invention based on the specific embodiments of the present invention.
In the examples of the present invention, all raw material components are commercially available products well known to those skilled in the art unless specified otherwise; in the embodiments of the present invention, unless specifically indicated, all technical means used are conventional means well known to those skilled in the art.
Example 1 acquisition of SNP loci closely related to aluminium tolerance of tomato
The method comprises the steps of planting by using 450 parts of tomato re-sequencing core germplasm materials, sampling, digesting, drying, grinding and aluminum ion determination aiming at tomato plants in a seedling stage, analyzing the tomato aluminum ion variation genetic basis by using whole genome association analysis, and detecting 1 obvious site on chromosome 12, wherein the specific reference can be seen in figures 1A and B. Analysis of 200kb upstream and downstream of this significant site found that one SNP was very significant at SL2.50ch12:1338779, see FIG. 1C.
Subsequently, we designed primers upstream and downstream of the site, respectively selected 3 extremely high materials and extremely low materials as templates for PCR, and found through sequencing that the extremely high materials have G to A mutation at the SNP site, see FIG. 1E, and further analyzed the genotype and aerial part aluminum ion determination results of all samples at the site, as shown in FIG. 1D, wherein the aerial part aluminum content of the material with the genotype AA is generally higher than that of the material with the genotype GA, and the aerial part aluminum content of the material with the genotype GA is generally higher than that of the material with the genotype GG. The gene sequence of the extremely high material is shown as SEQ ID NO. 1, and the gene sequence of the extremely low material is shown as SEQ ID NO. 2.
SEQ ID NO:1：CTATATAGTTTAAAGAGTTAAAAAAACATTTTATCA TTATTTTCGAAAGTTTCGTACCTTAACTATTCATTGTTCTCTTTTTTTACCCTCCATCACTTTGATGCTGAATTAGCCAAATATTTGTTTCCAATTGACTACAAACATGCGTACACTGATTCAACATCGAAGCGTGTTTTAATACTATAGTTATACCAGACCAACCTTTGTAGTATTTGCCTAGATGGATTCAGAATTTAATGTTTAAATATTTTGAACCACGACCTCTTTTCTATTTATTAAGTTTTGAATAGATACAAACAGATACAGAATTTGGACCAAAACTATTGAGTCATGACTTGTGTCAAAACTTGTAACTTCAACAGTAAATCCAGCCATATGCATACATCAATTGTATCAAAAAACAATAAAACTGTGATGTAAGTAAGTGCAACCAAATAACAGTTACATATCAAATGTAATCCGACAAGTGGAGTCTAAAGAAGGTAGAGTGTACGTAGACCTTACCCCGACCTCGTGGAGGTAGAGATAGAGAAACTGTTTCCGATACTTTGCGTTTACCTAGTGCAAGTGAGTGCAACCAAATGACTTATCTTTTTAAAAAGCCAAAAGAATAAGTAAATGGTAGTAGCAAGTCACTTAGCACAGCTTTGGGAAATAGTTGAAAATGTGAACATGATGCCTTGTGGCATCTCCCTATATACATCCATATTCCAAGACACAAAATCCTTACACTAAAGAAACAGGACATGCATGTTTCCTTTTTTTACTTCTTCACGAAATAAAAACCGCGTGAATGAGAACTAAAATGGTTCTTTCTATCGATCAAGATCAAATTAGCAAGAAACGAGACAGGTGGGGGGTTTAACGAGGCCTGGAATATTAGAAGGAACAGTAACAGGTTCATACTTGGCAGCAATTTCCAGACGCTCCCATGTCAACCTTCGTCTCTCCGCT；
SEQ ID NO:2：CTATATAGTTTAAAGAGTTAAAAAAACATTTTATCATTATTTTCGAAAGTTTCGTACCTTAACTATTCATTGTTCTCTTTTTTTACCCTCCATCACTTTGATGCTGAATTAGCCAAATATTTGTTTCCAATTGACTACAAACATGCGTACACTGATTCAACATCGAAGCGTGTTTTAATACTATAGTTATACCAGACCAACCTTTGTAGTATTTGCCTAGATGGATTCAGAATTTAATGTTTAAATATTTTGAACCACGACCTCTTTTCTATTTATTAAGTTTTGAATAGATACAAACAGATACAGAATTTGGACCAAAACTATTGAGTCATGACTTGTGTCAAAACTTGTAACTTCAACAGTAAATCCAGCCATATGCATACATCAATTGTATCAAAAAACAATAAAACTGTGATGTAAGTAAGTGCAACCAAATAACAGTTACATATCAAATGTAATCCGACAAGTGGAGTCTAGAGAAGGTAGAGTGTACGTAGACCTTACCCCGACCTCGTGGAGGTAGAGATAGAGAAACTGTTTCCGATACTTTGCGTTTACCTAGTGCAAGTGAGTGCAACCAAATGACTTATCTTTTTAAAAAGCCAAAAGAATAAGTAAATGGTAGTAGCAAGTCACTTAGCACAGCTTTGGGAAATAGTTGAAAATGTGAACATGATGCCTTGTGGCATCTCCCTATATACATCCATATTCCAAGACACAAAATCCTTACACTAAAGAAACAGGACATGCATGTTTCCTTTTTTTACTTCTTCACGAAATAAAAACCGCGTGAATGAGAACTAAAATGGTTCTTTCTATCGATCAAGATCAAATTAGCAAGAAACGAGACAGGTGGGGGGTTTAACGAGGCCTGGAATATTAGAAGGAACAGTAACAGGTTCATACTTGGCAGCAATTTCCAGACGCTCCCATGTCAACCTTCGTCTCTCCGCT。
According to the SNP locus, molecular marking type primer design is carried out, the sequence of a forward outer primer is shown as SEQ ID NO. 3, the sequence of a reverse outer primer is shown as SEQ ID NO. 4, the sequence of a forward inner primer is shown as SEQ ID NO. 5, and the sequence of a reverse inner primer is shown as SEQ ID NO. 6 in the designed ARMS-PCR primer.
Forward outer primer: 5'-TTTGCCTAGATGGATTCAGAATTTAATG-3' (SEQ ID NO: 3);
reverse outer primer: 5'-CAAAGCTGTGCTAAGTGACTTGCTACTA-3' (SEQ ID NO: 4);
forward inner primer: 5'-AATGTAATCCGACAAGTGGAGTCGAA-3' (SEQ ID NO: 5); reverse inner primer: 5'-GGGTAAGGTCTACGTACACTCTACCTTATC-3' (SEQ ID NO: 6).
The full length of amplification is 439bp, if the SNP locus has G to A mutation, 199bp and 439bp bands can appear, if no mutation exists, 295bp and 439bp bands can appear, if heterozygous, 199bp, 295bp and 439bp bands can appear, and the marker is named as AT12.
The amplified 199bp sequence is shown as SEQ ID NO. 7, and the amplified 295bp sequence is shown as SEQ ID NO. 8.
SEQ ID NO:7：AATGTAATCCGACAAGTGGAGTCTAAAGAAGGTAG AGTGTACGTAGACCTTACCCCGACCTCGTGGAGGTAGAGATAGAGAAACTGTTTCCGATACTTTGCGTTTACCTAGTGCAAGTGAGTGCAACCAAATGACTTATCTTTTTAAAAAGCCAAAAGAATAAGTAAATGGTAGTAGCAAGTCACTTAGCACAGCTTTG；
SEQ ID NO:8：TTTGCCTAGATGGATTCAGAATTTAATGTTTAAATAT TTTGAACCACGACCTCTTTTCTATTTATTAAGTTTTGAATAGATACAAACAGATACAGAATTTGGACCAAAACTATTGAGTCATGACTTGTGTCAAAACTTGTAACTTCAACAGTAAATCCAGCCATATGCATACATCAATTGTATCAAAAAACAATAAAACTGTGATGTAAGTAAGTGCAACCAAATAACAGTTACATATCAAATGTAATCCGACAAGTGGAGTCTAGAGAAGGTAGAGTGTACGTAGACCTTACCC。
Example 2
The embodiment provides an identification method of aluminum-resistant tomatoes, which utilizes the designed molecular marker primer to identify the screened extreme materials in tomato populations, and comprises the following specific steps:
extracting DNA of tomato material, using tomato gene DNA as template, the concentration is 80-120 ng/. Mu.L, using the molecular marker and primer in example 1 to amplify, wherein the total volume of PCR reaction is 20. Mu.L, the specific components are as follows: 2 xTag mix 9.6. Mu.L, outer primers 0.4. Mu.L each, inner primers 0.6. Mu.L each, DNA template 1. Mu.L (100-200 ng/. Mu.L), ddH 2 O 7.4μL。
The PCR reaction procedure was: pre-denaturation at 94℃for 3min, denaturation at 94℃for 30s, annealing at 55℃for 30s, extension at 72℃for 30s, 36 cycles, extension at 72℃for 5min, cooling to 15 ℃.
Gel electrophoresis detection: the PCR products were electrophoresed with 2% agarose at 100v for 40min and the final results were displayed on a gel imaging system.
Judgment of genotyping: the full length of amplification is 439bp, if the SNP locus has G to A mutation, 199bp and 439bp bands can appear, if no mutation exists, 295bp and 439bp bands can appear, and if the mutation exists, 199bp, 295bp and 439bp bands can appear, the corresponding aluminium intolerant genotype exists, and if the mutation exists at the same time, the bands can appear as heterozygous genotype.
Example 3
Four tomato materials TS-25, TS-181, TS-4, TS-9 (see Ye J, wang X, wang W, yu H, ai G, li C, sun P, wang X, li H, ouyang B, zhang J, zhang Y, han H, giovannoni JJ, fei Z, ye Z.genome-wide association study reveals the genetic architecture of 27agronomic traits in tomato.Plant Physiol.2021Aug3;186 (4): 2078-2092.Doi: 10.1093/pliys/kiab 230.PMID:34618111; PMCID: PMC 833593.) were selected for normal nutrient treatment for 28 days, and then normal nutrient and 50mM AlCl containing were performed, respectively 3 Nutrient solution treatment (ph=4.5 or so) for 10 days. The plant height and root length conditions of the four materials under different conditions are shown in figure 2, wherein 2C is a phenotype observation result, figure 2A is a plant height statistical structure, and figure 2B is a root length statistical result, and the figure shows that the plant height of the two materials TS-4 and TS-9 is obviously shortened under aluminum treatment, the plant height and root length of the two materials TS-25 and TS-181 are not obviously changed under aluminum treatment, and the two materials TS-4 and TS-9 are judged to be aluminum intolerant materials, and the two materials TS-25 and TS-181 are aluminum tolerant materials. Wherein the nutrient solution is as follows: 0.8mM Ca (NO 3 ) 2 ·4H 2 O，0.83mM KH 2 PO 4 ·3H 2 O，0.75mM MgSO 4 ·7H 2 O，1.5mM K NO 3 ，11.6μM H 3 BO 3 ，2.4μM MnSO 4 ·H 2 O，0.2μM ZnSO 4 7·H 2 O，0.1μM CuSO 4 ·5H 2 O，0.1μMNaMoO 4 ·2H 2 O，50μM FeSO 4 ·7H 2 O，50μM EDTA-Na 2 。
The DNA of the four materials is extracted, and detection is carried out according to the method described in the example 2, and the result shows that 295bp and 439bp bands appear in the two materials TS-4 and TS-9, 199bp and 439bp bands appear in the two materials TS-25 and TS-181, and the molecular marker detection result is consistent with the phenotype result, thus indicating that the molecular marker is used for accurately and reliably judging the materials of the aluminum-resistant tomatoes and the aluminum-intolerant tomatoes.
Example 4
180 parts of laboratory resequenced tomato TS population material (Ye et al, 2021) were selected, including 17 parts of mutant forms (TS-8, TS-35, TS-37, TS-41, TS-56, TS-131, TS-133, TS-154, TS-162, TS-181, TS-182, TS-184, TS-196, TS-198, TS-218, TS-222, TS-233), 161 parts of wild-type forms (TS-1, TS-2, TS-3, etc.), 2 parts of hybrid forms (TS-38, TS-105), the 180 parts of material described above were subjected to DNA extraction, PCR identification, and gel electrophoresis detection using the method of example 2, the analysis was performed by analyzing gel run typing results displayed by a gel electrophoresis imaging system, and the results showed that the molecular marker analysis results compare resequencing results, the percentage was calculated, and the marker identified tomato aluminium tolerance accuracy reached 89.4%. The mark has certain universality and accuracy, and can be used for screening the tomato aluminum-resistant material.
It should be noted that the above examples are only for further illustrating and describing the technical solution of the present invention, and are not intended to limit the technical solution of the present invention, and the method of the present invention is only a preferred embodiment and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The molecular marker AT12 for identifying the aluminium-tolerant gene type of tomatoes is characterized in that the molecular marker AT12 takes a tomato SL2.50 genome version as a reference gene, is positioned AT a 1338779 site on chromosome 12, and has G/A polymorphism.
2. A primer for amplifying the molecular marker AT12 of claim 1.
3. The primer for amplifying the molecular marker AT12 of claim 1 according to claim 2, wherein the primer comprises a forward outer primer as shown in SEQ ID NO. 3, a reverse outer primer as shown in SEQ ID NO. 4, a forward inner primer as shown in SEQ ID NO. 5, and a reverse inner primer as shown in SEQ ID NO. 6.
4. A kit for identifying an aluminium tolerance cause of tomato comprising the primer of claim 2 or 3.
5. A method for identifying an aluminium-tolerant cause of tomato comprising the steps of:
extracting total DNA of a tomato sample to be detected, amplifying by using the total DNA of the tomato to be detected as a template and a specific primer with a sequence shown as SEQ ID NO. 3-6, and judging according to the length of the amplified fragment;
the bands with the sizes of 199bp and 439bp are aluminum-resistant gene types, the bands with the sizes of 295bp and 439bp are aluminum-intolerant gene types, and the bands with the sizes of 199bp, 295bp and 439bp are heterozygous genotypes.
6. The method for identifying tomato aluminum-resistant genotypes as claimed in claim 5, wherein the system upon amplification is: 2 XTag mix 9.6. Mu.L, outer primers 0.4. Mu.L each, inner primers 0.6. Mu.L each, DNA template 1. Mu. L, ddH 2 O 7.4μL。
7. The method for identifying tomato aluminum-resistant genotypes as claimed in claim 5, wherein the procedure upon amplification is: pre-denaturation at 94℃for 3min, denaturation at 94℃for 30s, annealing at 55℃for 30s, extension at 72℃for 30s, 36 cycles, extension at 72℃for 5min, cooling to 15 ℃.
8. Use of the molecular marker of claim 1, the primer of claim 2 or 3 for identifying an aluminium tolerance cause of tomato.
9. Use of the molecular marker of claim 1, the primer of claim 2 or 3 in breeding aluminium-tolerant tomato varieties.
10. Use of the molecular marker of claim 1, the primer of claim 2 or 3 for cultivating aluminium-tolerant tomatoes.
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