CN118308510A - SNP locus related to mango anthracnose resistance, KASP (kalant-specific sequence repeat) labeled primer and application thereof - Google Patents
SNP locus related to mango anthracnose resistance, KASP (kalant-specific sequence repeat) labeled primer and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of mango breeding, and particularly relates to a SNP locus related to mango anthracnose resistance, a KASP (kalant-specific sequence repeat) labeled primer and application thereof. The SNP locus is positioned at 948977bp on chromosome 5 of mango, and the polymorphism of the RSME gene is A/G. The KASP mark primer of the SNP locus can be used as the auxiliary selection of mango anthracnose-resistant breeding marks, and provides a new molecular mark for cultivating genetically stable new anthracnose-resistant mango varieties.
Description
Technical Field
The invention belongs to the technical field of mango breeding, and particularly relates to a SNP locus related to mango anthracnose resistance, a KASP (kalant-specific sequence repeat) labeled primer and application thereof.
Background
Mango (MANGIFERA INDICA l.) is an important commercial fruit crop in tropical and subtropical areas, especially asia, widely planted in Guangdong and Guangxi Provinces, sichuan, taiwan, hainan and other tropical subtropical areas in our country.
The anthracnose (Colletotrichum gloeosporioides) of the mangiferum is one of important diseases in the production of the mangiferum, is caused by the anthracnose, and can infect mango leaves, inflorescences, fruits, branch tips and the like to cause necrosis of the branches and leaves, the inflorescences become black and dead, the bud fruits fall off and the like. In tropical and subtropical areas, the anthracnose of the mange is very rapid in development due to higher temperature and humidity, resulting in 30% -60% loss of fruits, even up to 100% loss in particularly humid environments. Disease-resistant breeding has been the most economical and effective way to deal with plant diseases.
The molecular markers linked with the mango anthracnose resistance genes are developed and screened out and used for detecting the disease resistance of different mango varieties (lines), an accurate and rapid research method can be provided for mango anthracnose resistance identification and mango disease resistance breeding, important requirements of sustainable development of mango industry in China are ensured, and important reference bases can be provided for molecular marker assisted selection and molecular breeding improvement of mango disease resistance.
Disclosure of Invention
Aiming at the problems, the invention discovers a SNP locus related to mango anthracnose resistance, designs a KASP (sequence-related amplified fragment) marker primer based on the locus, can accurately identify the sensitivity and resistance of mango anthracnose, and is beneficial to mango breeding.
In order to achieve the above purpose, the present invention may adopt the following technical scheme:
In one aspect, the invention provides a SNP locus related to mango anthracnose resistance, which is positioned at 948977bp of mango chromosome 5, and the polymorphism is A/G.
In another aspect, the invention provides a KASP-labeled primer for identifying SNP sites related to mango anthracnose resistance, which comprises a forward primer F1 with a sequence shown as SEQ ID NO.3, a forward primer F2 with a sequence shown as SEQ ID NO.4 and a universal reverse primer R with a sequence shown as SEQ ID NO. 5.
In a further aspect, the invention provides an identification reagent for identifying any one of the above SNP loci associated with mango anthracnose resistance, comprising a KASP-labeled primer as described in any one of the above.
In a further aspect, the invention provides an identification kit for identifying the SNP locus related to mango anthracnose resistance as set forth in any one of the above, which comprises the identification reagent as set forth in any one of the above.
In a further aspect, the invention provides an SNP locus related to mango anthracnose resistance as described above or a KASP (kalant-specific repeat) labeled primer as described above or an identification reagent as described above or an identification kit as described above, and application thereof in mango genetic breeding.
The beneficial effects of the invention at least comprise: based on the population verification, the KASP marker primer of the SNP locus related to mango anthracnose resistance reaches an extremely remarkable level according to the difference of the resistance reaction (disease index) between five types of phenotype five types of high resistance (3), medium resistance (4), medium sensitivity (4), disease (9) and high sensitivity (4) to anthracnose germs, and the developed KASP marker can divide 24 single plants into 3 types, disease-resistant varieties (14) and medium-sensitive varieties (1) and disease-sensitive varieties (9) and can effectively distinguish anti-sensitive materials from genotype level. The KASP markers developed above are applied to the collected mango varieties of 113 natural populations, and disease-resistant varieties (18), middle disease-sensitive varieties (50) and disease-sensitive varieties (45) are found; the SNP locus and the KASP marking primer provided by the invention can be used as auxiliary selection of mango anthracnose resistance breeding marks, and provide a new molecular mark for cultivating genetically stable new anthracnose resistance mango varieties.
Drawings
FIG. 1 is a chart of KASP markers developed versus 24 mango typing;
FIG. 2 is a graph of 113 natural population typing based on KASP markers.
Detailed Description
The examples are presented for better illustration of the invention, but the invention is not limited to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible in light of the above teachings and are intended to be within the scope of the invention.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. Unless the context clearly differs, singular forms of expression include plural forms of expression. As used herein, it is understood that terms such as "comprising," "having," "including," and the like are intended to indicate the presence of a feature, number, operation, component, part, element, material, or combination. The terms of the present invention are disclosed in the specification and are not intended to exclude the possibility that one or more other features, numbers, operations, components, elements, materials or combinations thereof may be present or added. As used herein, "/" may be interpreted as "and" or "as appropriate.
The embodiment of the invention provides a SNP locus related to mango anthracnose resistance, which is positioned at 948977bp of chromosome 5 of mango, and the polymorphism is A/G.
It should be noted that, according to the invention, 202 disease-resistant related KASP markers are obtained by carrying out gene function enrichment analysis screening through transcriptome and resequencing analysis of anthracnose high-resistance variety 'Jin Huang' and high-sensitivity variety 'Aivin', and according to the published 24 mango resistance phenotype identification results, one polymorphic site which is obviously related to mango anthracnose resistance is identified at 948977bp of chromosome 5, and exists in the coding Region of (RSME) ribosomal RNA small subunit methyltransferase E gene, guanine (G) is found in high-sensitivity material 'Aivin', and adenine (A) is mutated in high-resistance material 'Jin Huang'.
It should be noted that the gene annotated by the SNP site in the invention is RSME gene, which is a gene unified annotated by the SNP site related to the fight disease in combination with the information of the reference genome (https:// ngdc. Cncb. Ac. Cn/search/dbId = gwh & q= PRJCA 002248) of the mango variety 'red ivory'.
In some embodiments, the polymorphic site of the above SNP is located at the 251bp of the nucleotide sequence shown as SEQ ID NO.1 or SEQ ID NO. 2.
Specifically, the sequence of the disease-resistant material 'Jin Huang' carrying the KASP marker is shown as follows, wherein the 251 st position is the SNP site (KASP marker), and the polymorphism is "A";
GCATGCAAGTATACATTCTCAGCTCTCATACAAAAGCCACAATTTCAATTCCAGTCAAAGCCGCAACGTCATCGTATCGGTTCGATAGATAAAATGACCTCATTTTAAAAGCTGCATTTGAGGATCATTGGCATTTCCCAACTACATTCTTCATCAAGAGAAACTGGACGGCGAGACTTGGCACAGATGCGAAGCTTTGCGGCCGCAGTACGACCTTGTTTTGTTCAGCTGGTGAACCGTCCACCGTTTAAAATAAGAGCATTTTCTCGTTCATCCGATTACCCTAACCAATCACGCGGTTCTCTTCCTCGCTTCTTCTCCCAAGTTCTTCCTTCTTCCAAGGCAATCGATTTTCTATTTCCTTCAAGCCATGAACACATTTGTAAAATCATTCTTTTTTTAAAATTATTTTTTGGTGATAAATGGTGCAGGGAGGCGTTGTTCGCGTAGATGGAGATGAATTTTGGCATATGACTAAAGTCTTAAGGTTGACCACCAACG(SEQ ID NO.1).
The sequence of the susceptibility material 'Aiwen' carrying the KASP mark is shown as SEQ ID NO.2 (251 bp), wherein the 251 st position is the SNP locus (KASP mark), and the polymorphism is 'G';
GCATGCAAGTATACATTCTCAGCTCTCATACAAAAGCCACAATTTCAATTCCAGTCAAAGCCGCAACGTCATCGTATCGGTTCGATAGATAAAATGACCTCATTTTAAAAGCTGCATTTGAGGATCATTGGCATTTCCCAACTACATTCTTCATCAAGAGAAACTGGACGGCGAGACTTGGCACAGATGCGAAGCTTTGCGGCCGCAGTACGACCTTGTTTTGTTCAGCTGGTGAACCGTCCACCGTTTAGAATAAGAGCATTTTCTCGTTCATCCGATTACCCTAACCAATCACGCGGTTCTCTTCCTCGCTTCTTCTCCCAAGTTCTTCCTTCTTCCAAGGCAATCGATTTTCTATTTCCTTCAAGCCATGAACACATTTGTAAAATCATTCTTTTTTTAAAATTATTTTTTGGTGATAAATGGTGCAGGGAGGCGTTGTTCGCGTAGATGGAGATGAATTTTGGCATATGACTAAAGTCTTAAGGTTGACCACCAACG(SEQ ID NO.2);
another embodiment of the invention provides a KASP marker primer for identifying a SNP locus related to mango anthracnose resistance, which comprises a forward primer F1 with a sequence shown as SEQ ID No.3, a forward primer F2 with a sequence shown as SEQ ID No.4 and a universal reverse primer R with a sequence shown as SEQ ID No. 5.
Specifically, the KASP labeled primers are three primers, including forward primer F1, forward primer F2, and universal reverse primer R:
forward primer F1 (SEQ ID No. 3): TTAGCTAGTCCCACAGTCTTTG;
forward primer F2 (SEQ ID No. 4): TTAGCTAGTCCCACAGTCTTTA;
Universal reverse primer R (SEQ ID NO. 5): GGAAGAAGGAAGAACTTGGGAGAA.
In some embodiments, forward primer F1 and forward primer F2 in the KASP-labeled primer are each provided with a specific sequence that binds to a fluorescent label, and the bindable fluorescent labels are different.
In some embodiments, the specific sequence of the forward primer F1 in the KASP-labeled primer, which is combined with the fluorescent label, is shown in SEQ ID NO.6, and the specific sequence of the forward primer F2, which is combined with the fluorescent label, is shown in SEQ ID NO. 7.
Specifically, the 5 , end of forward primer F1 also includes a fluorescent label-binding specific sequence GAAGGTGACCAAGTTCATGCTGC (SEQ ID NO. 6), and specifically, the 5 , end of forward primer F2 also includes a fluorescent label-binding specific sequence GAAGGTCGGAGTCAACGGATTGC (SEQ ID NO. 7).
In some embodiments, the fluorescent label that binds to the specific sequence to which the forward primer F1 or F2 binds and the fluorescent label is known in the art, and may be selected according to the particular circumstances, such as the F1-linked may be HEX fluorescent label and the F2-linked may be FAM fluorescent label.
In yet another embodiment, the invention provides an identification reagent for identifying any of the above SNP loci associated with mango anthracnose resistance, comprising a KASP-labeled primer as described in any of the above. The KASP labeled primer described above may be added with an auxiliary reagent known in the art, such as a buffer, to prepare an identification reagent.
In yet another embodiment, the invention provides an identification kit for identifying any of the above SNP loci associated with mango anthracnose resistance, comprising any of the above identification reagents. It should be noted that the identification reagent may be prepared in a kit form, which is more convenient to transport and use, and the kit form is known in the art, for example, includes a reagent bottle, a specification, and the like.
The invention also provides an SNP locus related to mango anthracnose resistance or a KASP labeled primer or an identification reagent or an identification kit which are used in mango genetic breeding.
In some embodiments, the above-described applications include: if the genotype result of the mango to be identified is A/A, identifying the mango as an anthracnose resistance mango variety; if the genotype result of the mango to be identified is G/G, identifying the mango variety as anthracnose susceptibility mango variety; and if the genotype result of the mango to be identified is A/G, identifying the mango as the anthracnose inductive reactance neutral mango variety.
In some embodiments, the above-described applications include: the mass of the DNA of each sample to be tested for PCR reaction is between 100 and 1000ng, and the concentration of all the DNA to be tested should be uniform.
In some embodiments, the application may include:
(1) Extracting DNA of a sample to be detected;
(2) Performing PCR amplification on the DNA of the sample to be detected by using the KASP marked primer group to obtain an amplification product;
(3) Combining the amplified product with a fluorescent label, and detecting the fluorescent label;
Wherein, in the step (2), the PCR amplification reaction system may include: 1 mu L of DNA of a sample to be detected, PARMSMix mu L of primer group, 0.5 mu L of ddH 2 O1.5 mu L; the volume ratio of F1, F2 and universal reverse primer in the primers is 3:3:8.
Wherein, in the step (1), the PCR amplification reaction procedure is as follows: stage 1: pre-denaturation at 94℃for 15min; stage 2:94 ℃ for 20s and 65 ℃ for 1min, and the cycle is 10 times, and the cooling speed is-0.7 ℃/cycle; stage 3:94℃for 20s,57℃for 1min,28 cycles.
For a better understanding of the present invention, the content of the present invention is further elucidated below in connection with the specific examples, but the content of the present invention is not limited to the examples below.
Example 1
1. Sample to be measured
24 Mango samples to be tested were selected for identification, the information of which is shown in table 1 below.
TABLE 1 information on 24 mango samples to be tested
| Variety of infectious diseases | Disease resistant variety |
| Guiqi (Guiqi) | Shengxin |
| Aiwen (love) | White jade |
| Banana miscanthus | Gui heat 71 |
| Red mango No. 6 | Tiger head |
| Jade article | Thermonong No. 2 |
| Jin Suimang A | Sea ton |
| Gui Hui heat No. 10 | Aoan mango |
| Abutment tooth | Lv Song |
| Guba No. 1 | Yuexi No. one |
| Red ivory | Jin Huang A |
| 1506 | Incense with gold color |
| Imperial concubine | Apple tree |
2. Authentication process
(1) Kasp mark information
The KASP marker of mango anthracnose resistance gene is positioned at 948977 th base of mango chromosome 5, the base polymorphism is G/A, and the KASP marker is positioned in the coding Region of (RSME) ribosomal RNA small subunit methyltransferase E gene. The KASP of the anthracnose-resistant mango variety is marked as "A", and the KASP of the non-anthracnose-resistant mango variety is marked as "G".
(2) KASP labeled primer set
Forward primer f1+ fluorescent binding sequence:
GAAGGTGACCAAGTTCATGCTGCTTAGCTAGTCCCACAGTCTTTG；
forward primer f2+ fluorescent binding sequence:
GAAGGTCGGAGTCAACGGATTGCTTAGCTAGTCCCACAGTCTTTA；
universal reverse primer R: GGAAGAAGGAAGAACTTGGGAGAA.
(3) Extraction of leaf DNA
Extracting total DNA of 113 natural groups of leaves by adopting a CTAB method, which comprises the following steps: placing single young leaves into a 2mL centrifuge tube, adding two steel balls, 750uLCTAB extraction buffer solution (preheated at 60 ℃), grinding at 60HZ/90s by a grinder, standing at 65 ℃ for 30 minutes, slowly shaking uniformly for 1 time every 5 minutes, and completing DNA extraction by adopting a CTAB rapid extraction DNA kit; detecting the purity by 0.8% agarose gel electrophoresis and detecting the concentration by a spectrophotometer; finally, the mixture is diluted to a concentration of 50ng/uL and stored at-20 ℃ for later use.
(4) PCR and real-time quantitative fluorescence detection
The PCR reaction was 6. Mu.L, containing 1. Mu.L of the extracted sample DNA, 3. Mu. L PARMS Mix, 0.5. Mu. L PrimerMix (the volume ratio of F1, F2 and universal reverse primer in the primer set was 1:1:2) and 1.5. Mu.L of ddH 2 O;
The PCR reaction system is preheated at 94 ℃ for 15min; melting at 94 ℃ for 20s at 65 ℃ for 1min (-0.7 ℃/cycle), 10 cycles; 94℃20s,57℃1min,28 cycles; and at 35 ℃ for 1min.
The amplified product was read for endpoint signal using Applied BiosystemsTM QuantStudioTM and SNP-typed using its own software, black as negative control, red fluorescence for disease resistant genotype, green fluorescence for intermediate type, blue for disease sensitive type.
3. Experimental results
Through population verification, according to phenotype two types of disease resistance (12) and disease susceptibility (12), the difference of the resistance reaction (disease index) of anthracnose bacteria between the two types reaches an extremely significant level, the developed KASP markers can divide 24 single plants into 3 types, the disease resistance variety (14) and the middle disease susceptibility variety (1) and the disease susceptibility variety (9), the coincidence rate with the phenotype result is 75%, and the anti-susceptibility materials can be effectively distinguished from the genotype level.
Example 2
This example is intended to illustrate the use of the KASP marker in the identification of mango anthracnose resistance.
1. Sample to be measured
In the embodiment of the invention, 113 mango samples are selected for identification, the 113 mango samples are collected from Zhanjiang in Guangdong, the propagation modes are grafting, and the variety information is shown in Table 2.
TABLE 2 conditions of 113 mango sample varieties
2. Authentication process
(One) KASP flag information
A KASP marker of mango anthracnose resistance gene, wherein the KASP marker is positioned at 948,977 th base of mango chromosome 5, the base polymorphism is A/G, and the KASP marker is positioned in a coding Region of (RSME) ribosomal RNA small subunit methyltransferase E gene; the KASP of the anthracnose-resistant mango variety is marked as "A", and the KASP of the non-anthracnose-resistant mango variety is marked as "G".
(II) KASP labeled primer set
Forward primer f1+ fluorescent binding sequence:
GAAGGTGACCAAGTTCATGCTGCTTAGCTAGTCCCACAGTCTTTG
forward primer f2+ fluorescent binding sequence:
GAAGGTCGGAGTCAACGGATTGCTTAGCTAGTCCCACAGTCTTTA
Universal reverse primer R: GGAAGAAGGAAGAACTTGGGAGAA A
(III) extraction of leaf DNA
The total DNA of 113 natural groups of leaves is extracted by adopting a CTAB method. The method comprises the following steps: placing single young leaves into a 2mL centrifuge tube, adding two steel balls, 750uLCTAB extraction buffer solution (preheated at 60 ℃), grinding at 60HZ/90s by a grinder, standing at 65 ℃ for 30 minutes, slowly shaking uniformly for 1 time every 5 minutes, and completing DNA extraction by adopting a CTAB rapid extraction DNA kit; detecting the purity by 0.8% agarose gel electrophoresis and detecting the concentration by a spectrophotometer; finally, the mixture is diluted to a concentration of 50ng/uL and stored at-20 ℃ for later use.
Fourth, PCR and real-time quantitative fluorescence detection
The PCR reaction was 6. Mu.L, containing 1. Mu.L of the extracted sample DNA, 3. Mu. L PARMS Mix, 0.5. Mu. L PrimerMix (the volume ratio of F1, F2 and universal reverse primer in the primer set was 1:1:2) and 1.5. Mu.L of ddH 2 O.
The PCR reaction system is preheated at 94 ℃ for 15min; melting at 94 ℃ for 20s at 65 ℃ for 1min (-0.7 ℃/cycle), 10 cycles; 94℃20s,57℃1min,28 cycles; and at 35 ℃ for 1min.
The amplified product was read for endpoint signal using Applied BiosystemsTM QuantStudioTM and SNP-typed using its own software, black as negative control, red fluorescence for disease resistant genotype, green fluorescence for intermediate type, blue for disease sensitive type.
3. Experimental results
As shown in figure 2, the KASP marker provided by the invention is used for identifying anthracnose resistance of 113 natural population mango samples, and through population verification, the difference of resistance response (disease index) between four types of disease resistance types (24), middle resistance (25), middle feeling (24) and high feeling (40) of the phenotype reaches an extremely significant level, 113 single plants can be divided into 3 types by the developed KASP marker, the disease resistance type (18) and the middle-and middle-infected type (50) and the infected type (45) can be effectively distinguished from the genotype level according to the phenotype result coincidence rate of 58%.
Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.
Claims (10)
1. The SNP locus related to mango anthracnose resistance is characterized by being positioned at 948977bp of mango chromosome 5, and the polymorphism is A/G.
2. The SNP locus related to mango anthracnose resistance according to claim 1, wherein the polymorphic site of the SNP is located at the 251bp of the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO. 2.
3. A KASP marker primer for identifying the SNP locus associated with mango anthracnose resistance as defined in claim 1 or 2, comprising a forward primer F1 of sequence SEQ ID No.3, a forward primer F2 of sequence SEQ ID No.4 and a universal reverse primer R of sequence SEQ ID No. 5.
4. A KASP-labeled primer according to claim 3, wherein the forward primer F1 and the forward primer F2 are each provided with a specific sequence that binds to a fluorescent label and the bindable fluorescent labels are different.
5. The KASP-labeled primer according to claim 4, wherein the specific sequence of the forward primer F1 for binding to the fluorescent label is shown in SEQ ID NO.6, and the specific sequence of the forward primer F2 for binding to the fluorescent label is shown in SEQ ID NO. 7.
6. An identification agent for identifying SNP sites associated with mango anthracnose resistance as defined in claim 1 or 2, comprising a KASP marker primer as defined in any one of claims 3 to 5.
7. An identification kit for identifying the SNP locus associated with mango anthracnose resistance as defined in claim 1 or 2, comprising the identification reagent as defined in claim 6.
8. Use of a KASP marker primer according to any one of claims 3 to 5 or an identification reagent according to claim 6 or an identification kit according to claim 7 in mango genetic breeding for a SNP locus associated with mango anthracnose resistance according to claim 1 or 2.
9. The application of claim 8, the application comprising: if the genotype result of the mango to be identified is A/A, identifying the mango as an anthracnose resistance mango variety; if the genotype result of the mango to be identified is G/G, identifying the mango variety as anthracnose susceptibility mango variety; and if the genotype result of the mango to be identified is A/G, identifying the mango as the anthracnose inductive reactance neutral mango variety.
10. The use according to claim 8 or 9, wherein the mass of the sample DNA to be tested is between 100 and 1000ng per PCR reaction and the concentration of all the sample DNA to be tested should be uniform.
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| US20170327905A1 (en) * | 2016-05-13 | 2017-11-16 | Colorado State University Research Foundation | High throughput method to genotype plants |
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| US20170327905A1 (en) * | 2016-05-13 | 2017-11-16 | Colorado State University Research Foundation | High throughput method to genotype plants |
| KR101876273B1 (en) * | 2017-08-10 | 2018-07-09 | 순천대학교 산학협력단 | Molecular marker for selecting clubroot of Chinese cabbage and selection method using the same molecular marker |
| CN115747368A (en) * | 2022-10-20 | 2023-03-07 | 中国热带农业科学院南亚热带作物研究所 | KASP marker of mango bacterial angular leaf spot resistance gene and application |
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