CN114032330B - SNP molecular marker for rapidly identifying apple anthracnose - Google Patents
SNP molecular marker for rapidly identifying apple anthracnose Download PDFInfo
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Abstract
The invention belongs to the technical field of apple molecular breeding,relates to an SNP molecular marker for rapidly identifying apple anthracnose, wherein the SNP mutation site is positioned at 7309212bp of chromosome 15, and the mutation base and amino acid of the SNP mutation site are as follows: a is mutated to T; isoleucine is mutated to phenylalanine. The invention proposes to use the SNP 7309212 3 specific primers of the mutation sites can rapidly and accurately identify the resistance and the susceptibility of apples to anthracnose, the identification accuracy is 100%, and the accuracy of KASP typing technology is verified by an indoor leaf inoculation identification method. The invention provides an effective tool for early (seedling stage) auxiliary screening of new varieties of anthracnose-resistant apples by a molecular marking means, greatly improves the breeding efficiency and shortens the breeding period.
Description
Technical field:
the invention belongs to the technical field of apple molecular breeding, and in particular relates to an SNP molecular marker for rapidly identifying apple anthracnose by using a KASP typing technology, which can effectively carry out early disease resistance screening on apple hybrid seedlings and improve the efficiency of apple disease resistance breeding.
The background technology is as follows:
apple GLS is a disease which has serious influence on the development of apple industry in China in recent years, and mainly causes the dry and drop of leaves of main-cultivated varieties of apples such as 'Gala' and 'golden crown', causes necrotic lesions of fruits, causes the quality reduction of apples, and influences the next year production (Wang Bing, etc. 2014). Apple anthracnose originally occurred in the united states in 1970, but was not appreciated by people because the disease was not severe (Taylor et al, 1971). In 1988, the large-area explosion of GLS in the 'glara' apple cultivation garden in the south of brazil became a major disease causing serious economic loss of the brazil apple industry. The research and identification of Leite et al prove that the disease is named Glomerella leaf spot (Leite et al, 1988) and is named apple anthracnose in China.
Therefore, breeding of anthracnose-resistant varieties is imperative, and the auxiliary selection breeding by utilizing the traditional breeding combined with molecular markers is an effective means.
Molecular marker assisted selection breeding (Molecular marker-assisted selection, MAS) is the direct selection of phenotypes using Molecular markers closely linked to the trait of interest. SNP (Single nucleotide polymorphism), a single nucleotide polymorphism, is a polymorphism that results from a single nucleotide change at a certain point in two DNA sequences. The SNP marker has the advantages of large quantity, wide distribution and relatively stability, and can rapidly identify disease-resistant varieties and disease-sensitive varieties in early stages (seedling stages) through genotyping. Compared with the traditional breeding method of selecting genotype indirectly through phenotype, MAS breeding can effectively combine genotype and phenotype identification result, avoid blindness and unpredictability in the breeding process, thereby remarkably improving the selection accuracy and breeding efficiency.
KASP (Kompetitive Allele Specific PCR), i.e.competitive allele-specific PCR, a double allele determination was performed on the indicated SNPs (single nucleotide polymorphisms, single nucleotide polymorphism) and InDels (Insertions and Deletions ) based on specific matches of primer end bases. The KASP technology has low requirements on the purity and concentration of the detection sample (1-10 ng DNA can be added for reaction), and has the characteristics of high accuracy, flexibility and low cost. The specific expression supports low, medium and high flux sample research, and expensive specific fluorescent labeling primers or probes are not needed to be synthesized. The KASP technology has been widely used in agriculture and medicine, such as improvement of animal and plant species, disease screening and prediction, personalized medicine and prognosis, etc. For example, chinese patent CN201510807650.1 discloses a core SNP marker developed based on KASP technology for cotton hybrid identification, the SNP markers are distributed on 26 chromosomes of cotton tetraploid genome, each chromosome has 1SNP marker, and a total of 26 SNP markers, and based on the core SNP markers, high-throughput SNP typing detection of cotton hybrid can be realized; the invention discloses a primer combination for genotyping rice yield, which is developed based on a KASP technology, wherein the rice yield genes comprise Gn1a, NAL1, ghd7, hd1, ghd7.1 and Ghd8, the sequences of the primer combinations are respectively shown as SEQ ID NO. 1-18, the complete set of KASP functional markers and the primer combinations thereof provided by the invention can be used for rapidly detecting the combination mode of functional alleles of the yield genes in rice germplasm, the detection result is accurate and reliable, the operation is simple, the cost is low, the detection of important yield functional genes of rice breeding materials can be realized, and scientific guidance is provided for breeding and improving rice high-yield new varieties; chinese patent CN201810030833.0 discloses a set of core SNP markers for cabbage hybrid identification developed based on KASP technology, which comprise any one or more, or all of Bol01 to Bol50 SNP markers, and applications thereof; based on the core SNP marker, high-throughput SNP typing detection of cabbage hybrid can be realized. The rapid identification method and the used molecular marker related to the application are not found in the prior art, so that early disease resistance screening can be carried out on hybrid offspring in a seedling stage in the apple breeding process by using the method, and disease-resistant offspring plants are eliminated in the early stage, so that the disease resistance breeding efficiency of apples is greatly improved.
The invention comprises the following steps:
the invention aims to overcome the defects of the prior art, and seeks to provide a SNP molecular marker for rapidly identifying apple anthracnose by using a KASP typing technology, which can effectively carry out early disease resistance screening on apple hybrid seedlings and eliminate disease-sensitive offspring plants so as to achieve the purpose of improving the disease resistance breeding efficiency of apples.
In order to achieve the aim of the invention, the invention provides an SNP molecular marker for rapidly identifying apple anthracnose, wherein the SNP molecular mutation site is located at 7309212bp of chromosome 15.
The mutation bases and amino acids of the SNP mutation site are as follows: a is mutated to T; isoleucine to phenylalanine.
The types of SNP mutation sites are as follows: the bases of the disease-resistant tree body are heterozygous T/A and homozygous T/T, and the bases of the disease-resistant tree body are homozygous A/A.
The invention also provides a primer for rapidly identifying SNP molecular markers of apple anthracnose, wherein the specific primer for SNP loci of KASP reaction comprises a primer A1, a primer A2 and a primer C, and deoxyribonucleotide sequences of 3 primers are as follows:
primer A1: GAAGGTGACCAAGTTCATGCTCGAACTCCAATTTAATAAGTGATGCAA (SEQ ID NO. 1);
primer A2: GAAGGTCGGAGTCAACGGATTCGAACTCCAATTTAATAAGTGATGCAT (SEQ ID NO. 2);
primer C: ACTTCTAAAGAAGATATAGACCCGAGATC (SEQ ID NO. 3);
primer final concentration was 10. Mu.M, primer A1: primer A2: the primer C is mixed and added into the reaction system according to the volume ratio of 12:12:30.
The KASP reaction system of the present invention, comprising 5. Mu.l of 10 ng/. Mu.l DNA sample, 5. Mu.l of 2 XMaster mix and 0.14. Mu.l of mixed primer, was set up with no template control on each 96 well plate; the KASP reaction procedure was: the pre-denaturation stage lasts for 15min at 94 ℃; the denaturation stage lasts for 20s at 94 ℃, then the renaturation extension stage lasts for 60s at 61 ℃ for 10 cycles, and each cycle is reduced by 0.6 ℃; the final denaturation stage was carried out for 94℃for 20s,55℃for 60s and 26 cycles.
Compared with the prior art, the invention can simply and rapidly detect the typing condition of SNP loci by using KASP technology, further identify disease-resistant and disease-susceptible plants of apples, and is used for early disease-resistant identification and screening of hybrid seedlings in the apple breeding process.
Description of the drawings:
FIG. 1 shows the identification of susceptible and resistant plants at SNP by KASP typing technique according to example 2 of the present invention 7309212 Genotype profile of the site.
FIG. 2 is a graph showing the results of the disease resistance phenotype of different apple cultivars (lines) inoculated with C.fructicola according to example 3 of the present invention.
FIG. 3 is a graph showing the results of leaf plaque diameter after inoculation of C.fructicola with different apple varieties (lines) according to example 3 of the present invention.
The specific embodiment is as follows:
the invention will now be further illustrated by means of specific examples in connection with the accompanying drawings.
Example 1:
this example relates to a screening assay for rapidly identifying SNP molecular sites of apple anthracnose.
F of 'Jinguan' and 'Fuji' and 'Jinguan' X 'Fuji' apples planted in Jianzhou breeding base of Qingdao agricultural university in 2009 1 Carrying out Whole Genome Resequencing (WGR) on 20 strains of extreme resistance and 20 strains of extreme anthracnose-sensitive bacterial leaf blight in the generation population, using apple reference genome GDDH 13V 1, carrying out filtration screening on the obtained mutation sites, carrying out whole genome association analysis (GWAS) between disease-resistant and disease-sensitive populations by utilizing SNP markers obtained by filtration, and carrying out the strongest association signal on 17 chromosomes of the apple genome, wherein the 15 th chromosome shows the strongest association signal, and locking the SNP mutation site at 7309212bp of the 15 th chromosome of the apple genome by combining the published genetic map established in advance by the applicant. SNP (Single nucleotide polymorphism) 7309212 The mutation bases and amino acids of the mutation site are: a is mutated to T; isoleucine to phenylalanine. The types of mutation sites are: the bases of the disease-resistant tree body are heterozygous T/A and homozygous T/T, and the bases of the disease-resistant tree body are homozygous A/A.
Example 2:
the present example relates to the rapid identification of disease-susceptible and disease-resistant plants of different apple varieties (lines) using Kompetitive Allele Specific PCR (KASP) typing techniques, comprising the following specific steps:
(1) Selecting a test material:
the identification material was a total of 42 varieties (lines) of 26 individual plants and 16 varieties (table 1) produced and cultivated in the separated population of the 'Fuji' (disease resistance) and 'Jinguan' (disease resistance) hybrid F1 offspring of the breeding base of Qingdao agricultural university, which was planted in 2009.
(2) DNA extraction:
and extracting the DNA of the apple leaf sample to be identified by using a plant genome DNA extraction kit (Tiangen). DNA sample integrity was checked by 1% agarose gel electrophoresis and UV imager. The DNA concentration is 10-30 ng/. Mu.l, A260/280=1.8-2.0, A260/230=1.8-2.0.
(3) SNP using KASP technology 7309212 Genotyping at the locus:
obtained by previous screening by GWAS of applicantThe specific primer design is carried out on the obtained SNP molecular mutation site, so that the disease-resistant and disease-sensitive plants of the anthracnose are rapidly identified; the SNP mutation site is positioned at 7309212bp of chromosome 15 of the apple genome; SNP (Single nucleotide polymorphism) 7309212 The mutation bases and amino acids of the mutation site are: a is mutated to T; isoleucine to phenylalanine. The types of mutation sites are: the bases of the disease-resistant tree body are heterozygous T/A and homozygous T/T, and the bases of the disease-resistant tree body are homozygous A/A.
(4) Determining primer sequences: for SNP 7309212 The mutation site, 3 specific primers are designed for PCR reaction by using Primer 5 software, a GAAGGTGACCAAGTTCATGCT sequence connector is added to the 5 'end of the Primer A1, a GAAGGTCGGAGTCAACGGATT sequence connector is added to the 5' end of the Primer A2, and the sequence of the primers is as follows:
primer A1: GAAGGTGACCAAGTTCATGCTCGAACTCCAATTTAATAAGTGATGCAA (SEQ ID NO. 1);
primer A2: GAAGGTCGGAGTCAACGGATTCGAACTCCAATTTAATAAGTGATGCAT (SEQ ID NO. 2);
primer C: ACTTCTAAAGAAGATATAGACCCGAGATC (SEQ ID NO. 3).
SNP 7309212 Primers specific for the site were used at a final concentration of 10. Mu.M, primer A1: primer A2: the primer C is mixed and added into the reaction system according to the volume ratio of 12:12:30.
(5) Determining a reaction system: determination of the KASP reaction System was performed according to LGC company Kompetitive Allele Specific PCR (KASP) guidelines (www.lgcgenomics.com): the KASP reaction volume was 10.14. Mu.l, including 5. Mu.l of 10 ng/. Mu.l DNA sample, 5. Mu.l of 2 XMaster mix and 0.14. Mu.l of mixed primer, taking care that No Template Control (NTC) was placed on each 96-well plate.
(6) The KASP reaction procedure was determined as: the pre-denaturation stage lasts for 15min at 94 ℃; the denaturation stage lasts for 20s at 94 ℃, then the renaturation extension (61-55 ℃) stage lasts for 60s, 10 cycles are set, and the temperature is reduced by 0.6 ℃ per cycle; the final denaturation phase (94 ℃,20s,55 ℃,60 s) was continued for 26 cycles. The result data were analyzed using the Kraken software.
(7) Genotyping analysis: SNP on 42 apple samples Using KASP technique 7309212 The loci were genotyped and the results are shown in FIG. 1 and Table 1. Each dot in fig. 1 represents each sample to be tested. The results showed that SNP 7309212 There are 3 genotypes at the locus. In the absence of DNA template, the signal of NTC (black dots) as control was very weak, indicating that NTC did not cluster with other genotypes. 30 disease resistant plants with homozygous AA (blue dots) genotype were pooled together, 2 disease sensitive material with TT (red dots) genotype and 10 disease sensitive material with TA (green dots) genotype were pooled together (fig. 1, table 1). The samples to be tested of the blue dots are disease-resistant apple materials, and the samples to be tested of the red dots and the green dots are derived from the disease-resistant apple materials. As can be seen from the typing results, SNP 7309212 The parting effect of the locus in the apple anti-disease material is obvious, the disease-resistant plant and the disease-resistant plant can be obviously distinguished, and the identification efficiency can reach 100%.
TABLE 1SNP 7309212 Genotype distribution of F1 populations and cultivars of filial generation with loci in Fuji and golden crown
* The name of the DNA sample is preceded by "R" or "S" to indicate that the sample material is a "disease-resistant" or "disease-sensitive" material, and the numbers after R or S are line numbers.
Example 3:
the embodiment relates to the detection of the accuracy of KASP typing technology on the identification of anthracnose resistance of different apple varieties (systems) by using an indoor leaf inoculation identification method.
(1) Test materials and methods
In this example, two sets of hybrid offspring of the F1 population of the hybrid offspring of ' Fuji ' and ' Jinguan ' Fuji ' planted in the breeding base of Qingdao agricultural university in 2009 were respectively 26 and 10 and 21 cultivars were produced, and 57 varieties (lines) in total were used as test materials, and anthrax c. Structcola was inoculated to identify the disease and disease resistance of different apple varieties to anthrax leaf blight.
Culturing anthrax c. Fructicola strain and preparing suspension spore liquid:
the collected Ala' apple anthracnose leaf blight disease leaves are subjected to moisture preservation and culture for 3d in a room at 25 ℃, single spores are selected from conidia generated on the disease leaves, and are cultured in a PDA culture medium to obtain pure culture strains, and the pure culture strains are stored in a refrigerator at 5 ℃. Transferring the preserved strain into PDA culture medium before inoculation, activating at 25deg.C, scraping aerial hypha with inoculating loop when colony grows over 2/3 of culture dish, and continuing culturing at 25deg.C for 2-3 d until orange conidium angle grows out of culture medium.
Selecting proper amount of conidiophore with inoculating loop, shaking in beaker containing sterile distilled water, detecting spore suspension concentration with blood cell counting plate, and adjusting to 10 4 Individual mL -1 And (5) standby. The spore suspension is ready for preparation and the standing time is not more than 1h.
Determination of conidium germination force:
a drop of apple anthracnose spore suspension is dropped on a clean single concave glass slide, then a cover glass is covered, the apple anthracnose spore suspension is put into a culture dish with a small amount of water on the bottom surface, the dish cover is covered, and the apple anthracnose spore suspension is put into a culture box at 25 ℃ for culturing for 12 hours. The germination force of spores was observed under a microscope. The average germination force of spores is more than 20 percent.
The method for identifying the isolated inoculation comprises the following steps: annual robust shoots were cut from the apple tree tested, 4 shoots (2 for inoculation identification, 2 for control) were taken per material, both ends of the shoots were cut off, and the top 4 fully expanded leaves were retained. The surfaces of the leaves are disinfected by sodium hypochlorite of 0.6%, then washed by sterile water, drained, and the uniformly shaken conidium suspension is uniformly sprayed on the front and back surfaces of the leaves by a small sprayer until the leaves just start flowing water. Uniformly transplanting the inoculated and sterile distilled water sprayed branches (control) on two hole trays, placing the two hole trays in a foam box filled with a proper amount of distilled water, covering, sealing and moisturizing, and placing the two hole trays in a constant temperature incubator at 25 ℃ for dark culture. After 4 days, the identification of resistance and data recording were performed, and the leaf was marked as "disease-resistant" and "disease-sensitive" and "disease-free" and "disease-sensitive" were marked as (S). Chi-square test analysis was performed using SPSS13.0 software.
(2) Test results
The results of leaf disease are shown in Table 2, FIG. 2 and FIG. 3. Statistical analysis of leaf spot areas by a crisscross method is adopted, and disease resistance division standards of apple germplasm resources are adopted (Wu Jianyuan and the like, 2017).
From the results of the in vitro inoculation identification of the leaves, the identification result by using the KASP typing technology is completely consistent with the result of the indoor inoculation of pathogenic bacteria of the leaves, and the method for identifying the anthracnose resistance of the apples by using the KASP typing technology is proved to be effective.
TABLE 2 statistics of resistance of different apple varieties (lines) after in vitro C.fructicola inoculation of leaves
Example 4:
this example relates to a control method of the KASP typing technique: common PCR amplification method of SNP mutation site identifies anthracnose resistance of different apple varieties (lines).
To compare the efficiency and accuracy of KASP typing technology in identifying anthracnose resistance, SNP was used 7309212 PCR amplification method of mutation site identifies anthracnose resistance of different apple varieties.
(1) Test materials and methods
In this example, 21 and 13 hybrid offspring F1 populations of "Fuji" and "Jinguan" x "Fuji" planted in the breeding base of Qingdao agricultural university in 2009 were produced from 21 cultivars, and a total of 55 cultivars were producedThe strain (line) is used as a test material, leaf DNA is extracted as a template, an autonomously designed specific primer (SNP-F: AGATTGATACTCAGAGACTG (SEQ ID NO. 4)) is used as a reaction primer, and a PCR amplification program with a reaction system of 50 μl is set. The reaction system included 25. Mu. l Accurate Taq enzyme, 2. Mu.l template, 2. Mu.l each SNP-F/R primer and 19. Mu.l ddH2O. Mixing the samples uniformly, and placing the mixture into a PCR instrument for amplification, wherein the amplification procedure is as follows: pre-denaturation at 95 ℃ for 5min; the cycle times of denaturation (95 ℃,30 s), annealing (55 ℃,30 s) and extension (72 ℃,30 s) stages are 35; and finally, storing at 72 ℃,10min and 4 ℃. After the PCR is finished, checking the integrity of the target band by 1% agarose gel electrophoresis, selecting the reaction solution with the intact band, sending to Shanghai biological company Limited for sequencing, and finally comparing SNP 7309212 Sequencing results and peak plots.
(2) Test results
SNP (Single nucleotide polymorphism) for obtaining 55 different apple disease resistance samples through PCR (polymerase chain reaction) 7309212 The sequencing results are shown in Table 3. The results in Table 3 show that of the 55 apple varieties (lines), 37 samples were AA (single peak AT mutation site, A as sequencing base result, and homozygous AA as judgment), 18 samples were AT (double peak AT mutation site, A and T as sequencing base result, and heterozygous AT as judgment), and according to the previous research results of applicant, AA as genotype was disease resistant variety (line), and AT as genotype was disease sensitive material. According to leaf inoculation experiments and field observations, 3 samples have errors, namely the amplified genotype is inconsistent with the anti-disease result of the actual variety, 3 samples are respectively the Lu Jia No.1 disease-resistant variety, the genotype is AA, and the genotype amplified by the method is AT;13-26 are disease-resistant varieties, the genotype should be AA, and the genotype amplified by the method is AT; double yang red is a disease-sensitive variety, the genotype should be AT, and the genotype amplified by the method is AA.
From the above results, it can be seen that 3 out of 55 samples were erroneous in identifying the anthracnose resistance of different apple varieties (lines) by PCR amplification method using SNP mutation sites, the identification accuracy was 94.5%, and the identification accuracy was inferior to KASP typing technique.
Table 3 different apple productsSeed (line) SNP 7309212 Genotype of mutation site:
note that: the mutation site of the sequencing peak diagram is unimodal, the sequencing base result is A, and the genotype is judged to be homozygous AA; the mutation site of the sequencing peak diagram is bimodal, the sequencing base results are A and T, and the genotype is judged to be heterozygous AT.
The application result of the embodiment shows that the technique can be used for rapidly identifying the early apple anthracnose resistance of the hybrid seedlings of the apples, has high identification speed and high identification accuracy, greatly saves the breeding time, improves the breeding efficiency and is environment-friendly to application.
Sequence listing
<110> Qingdao university of agriculture
<120> SNP molecular marker for rapidly identifying apple anthracnose
<130> 2021
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Claims (3)
1. The primer for rapidly identifying the SNP molecular marker of the apple anthracnose is characterized in that a SNP molecular mutation site is positioned at 7309212bp of chromosome 15, and the mutation base and amino acid of the SNP mutation site are as follows: a is mutated to T; isoleucine to phenylalanine; the types of SNP mutation sites are: the bases of the disease-resistant tree body are heterozygous T/A and homozygous T/T, and the bases of the disease-resistant tree body are homozygous A/A; specific primers for SNP sites used in the KASP reaction include primer A1, primer A2 and primer C, and the deoxyribonucleotide sequences of the 3 primers are:
primer A1: GAAGGTGACCAAGTTCATGCTCGAACTCCAATTTAATAAGTGATGCAA;
primer A2: GAAGGTCGGAGTCAACGGATTCGAACTCCAATTTAATAAGTGATGCAT;
primer C: ACTTCTAAAGAAGATATAGACCCGAGATC.
2. The primer for rapidly identifying SNP molecular markers of apple anthracnose according to claim 1, wherein the final primer concentration is 10. Mu.M, primer A1: primer A2: the primer C is mixed and added into the reaction system according to the volume ratio of 12:12:30.
3. The primer for rapidly identifying SNP molecular markers for apple anthracnose according to claim 1, wherein the KASP reaction system comprises 5 μl 10ng/μl DNA sample, 5 μl 2 x Master mix and 0.14 μl mixed primer, and no template control is set on each 96 well plate; the KASP reaction procedure was: the pre-denaturation stage lasts for 15min at 94 ℃; the denaturation stage lasts for 20s at 94 ℃, then the renaturation extension stage lasts for 60s at 61 ℃ for 10 cycles, and each cycle is reduced by 0.6 ℃; the final denaturation stage was carried out for 94℃for 20s,55℃for 60s and 26 cycles.
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