Molecular marker for detecting chilling requirement trait of pear and application thereof

Abstract

The application discloses a kind of molecular markers for detecting pear chilling requirement character and its application, belong to plant molecular marker technical field.The molecular marker is the single nucleotide polymorphism C>A of DAM1 gene CDS region shown in SEQ ID NO.1 216th position, utilize the SNP as detection target point and be applied to detect pear chilling requirement character.The application first discloses that the SNP is significantly associated with pear low chilling requirement character, by detecting the single nucleotide polymorphism of this site, the chilling requirement character of different pear varieties can be effectively identified, which has important significance for low chilling requirement pear variety identification and new variety breeding, shortening breeding period and improving breeding efficiency.The application also provides a method for detecting low chilling requirement pear variety / strain using KASP technology, which can efficiently detect the single nucleotide polymorphism of specific gene site of test variety / hybrid offspring.The method is fast, simple, specific and can realize high-throughput detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant molecular markers, and particularly relates to a genetic molecular marker for detecting a chilling requirement trait of a pear and application thereof. BACKGROUND

[0002] Pear is a deciduous fruit tree crop, which originated from high-altitude areas near the Hengduan Mountains in Southwest China. After long-term natural selection and human domestication, it has become the most widely cultivated temperate fruit tree crop in China, and a series of local varieties have been formed to adapt to the local cultivation climate environment in various parts of the country.

[0003] During the long evolution process, pear has formed a natural bud dormancy biological process to cope with the change of cold climate in winter. When the pear bud enters natural dormancy, it cannot resume growth even in a suitable growth environment, and must be exposed to low temperature for a long time to naturally break dormancy. The low temperature duration (chilling requirement) required to break dormancy varies greatly among different varieties. The chilling requirement of different varieties is closely related to their environmental adaptability. Varieties originating from South China generally exhibit low chilling requirement.

[0004] In recent years, with the intensification of global climate change, the duration of low temperature in winter in many regions of China has become shorter, and the original suitable varieties have shown insufficient dormancy release in winter. Therefore, local varieties originating from southern China, as an important source of low chilling requirement, have attracted attention. However, how to use molecular markers to distinguish low chilling requirement varieties in southern China has not been reported.

[0005] DAM (Dormancy Associated MADS-box) protein is a key transcription factor regulating dormancy in woody plants, belonging to the AGL24 / SVP branch of the MADS-box family. In pear, five DAM genes have been cloned, located on chromosomes 8 and 15, and showing tandem duplication on chromosome 8. During the internal dormancy process of ‘Dangshan pear’ buds, the expression levels of the five DAM genes all changed, and their expression levels increased with the deepening of dormancy, and rapidly decreased after dormancy release (Gao et al, 2021). However, the relationship between the variation of DAM genes and the geographical distribution of varieties has not been reported.

[0006] With the development of plant science, plant variety identification has entered the gene level. Compared with the first generation of RFLP and the second generation of BSA, single nucleotide polymorphism (SNP) as the third generation of molecular marker technology has many advantages such as high throughput, low cost and easy operation, and is the main position of future molecular markers and has great prospects. Competitive allele specific PCR (KASP) is a new SNP-based genotyping technology, which can accurately determine the double alleles of SNPs and InDels at specific sites in a wide range of genomic DNA samples (even some complex genomic DNA samples). Since pear is a strict self-pollinating variety, most varieties are artificially propagated by bud mutation selection, and the genomes of different varieties are extremely similar, and the genetic background is single. At present, there are few molecular markers for identifying pear traits. Therefore, the development of KASP molecular markers for pear has important significance for variety identification and new variety breeding. SUMMARY

[0007] The purpose of the present application is to provide a genetic molecular marker for identifying the chilling requirement trait of pear, which can distinguish low chilling requirement pear varieties at low cost, so as to adapt to the areas with shorter winter low temperature.

[0008] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0009] The present application provides the application of a single nucleotide polymorphism (SNP) site as a detection target in the preparation of a kit for detecting the chilling requirement trait of pear, wherein the single nucleotide polymorphism site is a C>A polymorphism at position 216 of the nucleotide sequence shown in SEQ ID NO. 1.

[0010] The present application compares the DAM gene locus sequences of pear varieties with generally low chilling requirement and high chilling requirement through genome resequencing, and finds that there is a SNP site with base substitution at position 216 of the CDS region (the nucleotide sequence is shown in SEQ ID NO. 1) of DAM1 gene (NCBI accession number XM_009377956.3) of the low chilling requirement pear variety, and the nucleotide mutation type of the position is C→A. The mutation site is located at position 18682690 of chromosome 8 of 'Yali' genome.

[0011] By analyzing the genotype of the above-mentioned site in a large sample of pear, the base at position 216 of the CDS region of DAM1 gene includes two states of base C and base A, and the corresponding genotype is CC and CA.

[0012] The research shows that the base variation of C>A is significantly related to the low chilling requirement trait. When the base is C, it is the normal genetic state of DAM1 gene, and when the base is A, a stop codon TAA is formed, which leads to the premature termination of protein translation, so that the formed protein lacks the subsequent active domain, resulting in protein inactivation, and the plant shows low chilling requirement trait. Compared with the spring flowering time of individuals with genotype CC, the spring flowering time of individuals with CA genotype is significantly advanced, corresponding to the low chilling requirement trait.

[0013] Therefore, the above-mentioned SNP can be used as a molecular marker for identifying pear varieties with low chilling requirement trait, or a genetic molecular marker for determining the chilling requirement of offspring when using pear varieties with low chilling requirement trait as parents for variety breeding.

[0014] As an application of the above-mentioned molecular marker, the present application provides a kit for detecting the chilling requirement trait of pear, which comprises: specific primers for detecting the single nucleotide polymorphism at position 216 of the nucleotide sequence shown in SEQ ID NO. 1.

[0015] The specific primers are two pairs, and two specific upstream primers and one specific downstream primer are designed for the single nucleotide polymorphism of the specific site of the DAM gene. Each specific upstream primer corresponds to the corresponding C and A base sequence, respectively, and is specifically amplified with the downstream primer by PCR, and the genotype of the corresponding base is determined to be homozygous or heterozygous according to the PCR positive band amplified by the corresponding primer pair.

[0016] Further, the sequences of the upstream and downstream primers of primer pair I are respectively:

[0017] The upstream primer is 5'-TACCAAGGATGTGATTGCAAGGTAC-3' (SEQ ID NO. 2);

[0018] The downstream primer is 5'-ATTTGATCCGATTTTTCCCCACCAGTATG-3' (SEQ ID NO. 4);

[0019] The sequences of the upstream and downstream primers of primer pair II are respectively:

[0020] The upstream primer is 5'-TACCAAGGATGTGATTGCAAGGTAA-3' (SEQ ID NO. 3);

[0021] The downstream primer is 5'-ATTTGATCCGATTTTTCCCCACCAGTATG-3' (SEQ ID NO. 4).

[0022] The application also provides a KASP kit for detecting the chilling requirement trait of pear, which comprises a primer set for specifically detecting the single nucleotide polymorphism at position 216 in the nucleotide sequence shown in SEQ ID NO. 1.

[0023] Specifically, the primer set comprises two upstream typing primers with different universal fluorescent linker sequences and one downstream universal primer. The KASP reaction system contains two fluorescent probes and two complementary quenched probes, and the fluorescent probe is consistent with the universal fluorescent linker sequence of the upstream typing primer. With PCR amplification, the universal fluorescent linker sequence is introduced into the PCR product corresponding to the SNP, the fluorescent probe is added to the PCR product by binding to the complementary DNA strand of the universal fluorescent linker sequence, and the quenched probe that is no longer complementary to it is combined, thereby generating a fluorescence signal. After the reaction is completed, the fluorescence intensity of two different wavelengths in the reaction solution is read to determine the genotype of the corresponding base.

[0024] Further, the KASP reaction primer set sequence is as follows:

[0025] The upstream typing primer F1 is 5'-GAAGGTGACCAAGTTCATGCTTACCAAGGATGTGATTGCAAGGTAC-3' (SEQ ID NO. 5);

[0026] The upstream typing primer F2 is 5'-GAAGGTCGGAGTCAACGGATTTACCAAGGATGTGATTGCAAGGTAA-3' (SEQ ID NO. 6);

[0027] The downstream universal primer R is 5'-ATTTGATCCGATTTTTCCCCACCAGTATG-3' (SEQ ID NO. 4).

[0028] Further, the kit also comprises reagents such as PCR Premix required for KASP reaction, and a commercially available conventional KASP marker kit can be used instead, which is conventional knowledge for those skilled in the art.

[0029] As an application of the above kit, the application also provides a method for identifying a pear variety / strain with low chilling requirement, comprising the following steps:

[0030] (1) Extracting the genomic DNA of the pear to be tested as a template, and performing PCR reaction by using the primer set for specifically detecting the single nucleotide polymorphism at position 216 in the nucleotide sequence shown in SEQ ID NO. 1;

[0031] (2) Determining the chilling requirement trait of the sample to be tested according to the genotype of the product:

[0032] If the product genotype is CA, the sample to be tested is judged as a low cold requirement variety;

[0033] If the product genotype is CC, the sample to be tested is judged as a high cold requirement variety.

[0034] When the pear individual cold requirement trait is analyzed by the above method, if the normal DAM1 gene base is compared, and if there is a difference, that is, single nucleotide polymorphism: 216C>A, it indicates that the individual is a low cold requirement variety, which is suitable for cultivation in the warm southern winter area.

[0035] Further, the KSAP technology is used to identify the cold requirement trait of pear, specifically, in step (1), the primer group with the nucleotide sequence shown in SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 4 is used for KASP reaction detection; in step (2), the reaction product is read for fluorescence data, and each sample is genotyped according to the relative intensity of different wavelengths of fluorescence:

[0036] If the fluorescence is orange yellow, the genotype is CC;

[0037] If the fluorescence is green, the genotype is CA.

[0038] Further, the composition of the KASP reaction system includes: 1x KASP premix, the final concentration of the upstream typing primer F1 is 0.1 μM, the final concentration of the upstream typing primer F2 is 0.1 μM, the final concentration of the downstream universal primer R is 0.2 μM, and the genomic DNA template is 10~50 ng, with a total volume of 10 μL.

[0039] Further, the KASP reaction conditions are: 95℃ pre-denaturation for 10 min, 1 cycle; 95℃ denaturation for 15 s, 61~55℃ annealing and extension for 60 s, 10 cycles, and the temperature of annealing and extension decreases by 0.6℃ for each cycle; 95℃ denaturation for 15 s, 55℃ annealing and extension for 60 s, 28~35 cycles.

[0040] Compared with the prior art, the present application has the following advantages:

[0041] (1) The present application first discloses that the single nucleotide polymorphism existing in the 216th position of the CDS region of DAM1 gene is significantly related to the low cold requirement trait of pear, and by detecting the single nucleotide polymorphism of the site, the cold requirement trait of different pear varieties can be effectively identified. This has important significance for identifying low cold requirement pear varieties, breeding new varieties, shortening the breeding period and improving the breeding efficiency.

[0042] (2) The application provides a kit for detecting low chilling requirement pear varieties / lines, wherein the kit contains three KASP primers designed based on DAM1 single nucleotide polymorphism. The KASP technology can be used to efficiently detect the single nucleotide polymorphism of a specific gene site of a test variety / hybrid offspring, and the method is rapid, simple, specific and can realize high-throughput detection. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 A mutation schematic diagram of a DAM1 gene variation site.

[0044] Figure 2 A result analysis diagram of KASP typing using 19 pear materials, wherein a regular triangle represents a genotype CC sample (homozygous type), and a circle represents a genotype AC sample (heterozygous type).

[0045] Figure 3 It is the identification result of four pear chilling requirement genotypes by using Sanger sequencing method. DETAILED DESCRIPTION

[0046] The application will be further described below in combination with specific examples. The following examples are only used to illustrate the application, and are not used to limit the application scope. Modifications or replacements of the application method, steps or conditions without departing from the spirit and essence of the application all belong to the scope of the application.

[0047] The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used are reagents and materials that can be obtained through commercial channels unless otherwise specified.

[0048] Example 1

[0049] 1. DAM1 gene nucleotide polymorphism (SNP) site analysis

[0050] In this study, the genome resequencing (about 10X) data of 9 local pear varieties such as 'Yeshen pear', 'Nanning big sand pear', 'Taiwan green flower' and 12 varieties such as 'Dangshan crisp', 'Cui Guan', 'duck pear','red early crisp' in South China such as Fujian, Guangdong and Guangxi were obtained, and the single nucleotide polymorphism and InDel variation in the DAM gene locus sequence of these varieties were compared, and the reference genome of 'Huaershan' was used as a reference to obtain the single nucleotide polymorphism (SNP) site of DAM1 gene. Figure 1A SNP site of base substitution was found at the 216th base of the CDS region of DAM1 originated from pear varieties in South China, and the nucleotide mutation type of the position was C→A. When the base is C, it is the normal genetic state of DAM1 gene, and when the base is A, a stop codon TAA is formed, which leads to premature termination of protein translation, so that the formed protein only has part of the MADS-box domain, but lacks the subsequent K-box and other active domains, resulting in inactivation of the protein function.

[0051] The nucleotide sequence of the CDS region of DAM1 gene is shown in SEQ ID NO. 1.

[0052] 2. Association analysis of nucleotide polymorphism of DAM1 gene and chilling requirement trait

[0053] The SNP genotypes of the site in 264 pear samples from different regions in China were analyzed by genome resequencing, and it was found that there were CC and CA types, and no pear variety with AA type was found.

[0054] It is known that the early flowering time in spring has a significant correlation with the chilling requirement trait, and the early flowering time in spring is generally used to represent the relative relationship of chilling requirement in the study (Castede et al, 2014). The flowering time of the first variety in the population was used as zero point, and the difference between the flowering time of the remaining varieties and the zero point was calculated. The early flowering time in spring of the above-mentioned 264 samples was further analyzed, and it was found that the SNP site had a significant correlation with the early flowering time in spring of the varieties, and the results are shown in Table 1.

[0055] Table 1. Correlation between DAM1 base variation and early flowering time in spring

[0056]

[0057] As can be seen from Table 1, the early flowering time in spring of the individuals with CA genotype is significantly advanced by about 6 days, corresponding to low chilling requirement trait, and the varieties determined as CA type include ‘Yeshen pear’, ‘Nanning large sand pear’, ‘Taiwan green flower’, ‘Taiwan red flower’, ‘Hengxian sour pear’, ‘Hengxian large heart pear’, ‘Huizhang pear’, ‘E pear’ and ‘Chapip pear’.

[0058] Therefore, the above-mentioned SNP can be used as a molecular marker for the low chilling requirement trait of the above-mentioned varieties, or as a genetic molecular marker for determining the chilling requirement of the offspring when using the above-mentioned varieties as parents for variety breeding.

[0059] Example 2

[0060] 1. Based on the results of Example 1, we found that the above-mentioned base variation of C>A was significantly correlated with the low chilling requirement trait. Therefore, we designed KASP marker primers for detecting single nucleotide polymorphism at specific sites of the DAM1 gene, which consisted of two specific upstream primers and one specific downstream primer, as follows:

[0061] Forward primer F1: 5'-GAAGGTGACCAAGTTCATGCTTACCAAGGATGTG ATTGCAAGGTAC-3';

[0062] Forward primer F2: 5'-GAAGGTCGGAGTCAACGGATTTACCAAGGATGT GATTGCAAGGTAA-3';

[0063] Reverse primer R: 5'-ATTTGATCCGATTTTTCCCCACCAGTATG-3';

[0064] Each specific upstream primer corresponds to the corresponding C or A base sequence, respectively, and is specifically amplified with the downstream primer by PCR, and the length of the amplification product is 60 bp.

[0065] The front end of forward primer 1 is labeled with a universal fluorescent sequence FAM, and the front end of forward primer 2 is labeled with a universal fluorescent sequence VIC. After the reaction is completed, the fluorescence intensities of the two different wavelengths in the reaction solution are read to determine the genotype of the corresponding base.

[0066] 2. Identification of genotypes of different pear varieties at specific sites of the DAM1 gene using KASP marker primers for PCR amplification

[0067] 2.1 There were 19 test pear varieties, specifically: 'Chapipir', 'Nanning Dasha pear', 'Huizhang pear', 'Yeshen pear', 'Hengxian Daxin pear', 'Taiwan Qinghua', 'Nanjing Chapi pear', 'Zhangpu Chapi pear', 'Hengxian Se pear', 'Taiwan Chihua', 'Guihua', 'Hengshan', 'Nanjing Chixin pear', 'Qinghua pear', 'Chihua pear', 'Huizhang sour pear', 'E pear', 'Dangshan Kuo', and 'Hongzaokuo'.

[0068] Among them, 'Yeshen pear', 'Nanning Dasha pear', 'Taiwan Qinghua', 'Taiwan Chihua', 'Hengxian Se pear', 'Hengxian Daxin pear', 'Huizhang sour pear', 'E pear', and 'Chapipir' are 9 varieties that have been determined by genome resequencing to be CA type varieties;

[0069] 'Nanjing red skin pear', 'Zhangpu red skin pear', 'Huizhang sand pear', 'Guihua', 'Hengshan', 'Nanjing red heart pear','red flower pear' and 'green flower pear' are pear varieties collected from Fujian, Guangdong and Guangxi, etc. They can adapt to the climate and environment of South China and can normally blossom and bear fruit under the environment of less than 300 hours of 0-7.2°C low temperature accumulation in winter in the original place, being low cold requirement varieties.

[0070] 'Dangshan Qiu' and 'Hongzaqiu' are typical northern pear varieties. Previous studies have shown that the 0-7.2°C cold requirement of 'Dangshan Qiu' is more than 1230 hours (Feng Lei et al., 2013), being a high cold requirement variety.

[0071] 2.2 Extraction of genomic DNA: 0.2 g of young tissue of pear tree was taken, and the traditional CATB method or commercially available plant genomic extraction kit was used for extraction of genomic DNA. The extracted genomic DNA was dissolved in deionized water, and the concentration was adjusted to about 100 ng / μL, and was stored at low temperature.

[0072] 2.3 Composition of PCR reaction system: with a total volume of 10 μL, containing FLu-arms 2x PCR Mix 5 μL, 10 μM forward primer F1 0.1 μL, 10 μM forward primer F2 0.1 μL, 10 μM reverse primer 0.2 μL, and 20 ng of genomic DNA template.

[0073] When performing genotype detection, KASP genotyping mixed solution was prepared according to the above formula (except for DNA template), 50 portions of mixed solution were prepared, and KASP genotyping mixed solution was sucked and beaten with a pipette gun and centrifuged, then was divided into 96-well plates, 10 μL per well, 20 ng of DNA template was added to each well, and each variety was repeated twice. The PCR plate was sealed with a sealing film, and then was centrifuged.

[0074] 2.4 PCR amplification: PCR amplification was performed using a general PCR instrument with a touchdown PCR program, and the amplification program is shown in Table 2.

[0075] Table 2. PCR amplification program

[0076]

[0077] After the completion of PCR amplification, the 96-well plate was taken out, and the fluorescence was read using a fluorescent quantitative PCR instrument (Bio-Rad), and the reading program was set as: 30°C for 5s+plate read. The fluorescence types FAM, HEX (i.e. 550nm channel, suitable for VIC marker) were selected.

[0078] 2.5 Cluster typing of all test varieties according to the nucleotide types of the SNP sites.

[0079] As Figure 2 shown, 19 test pear varieties were clustered into two different types, wherein ‘Chipi pear’, ‘Nanning large sand pear’, ‘Huizhang pear’, ‘Yeshen pear’, ‘Hengxian large heart pear’, ‘Taiwan green flower’, ‘Nanjing Chipi pear’, ‘Zhangpu Chipi pear’, ‘Hengxian astringent pear’, ‘Taiwan Chihua pear’, ‘Guifahua pear’, ‘Hengshan pear’, ‘Nanjing Chixin pear’, ‘Qinghuapir’, ‘Chihua pear’, ‘Huizhang sour pear’, and ‘E pear’ showed CA heterozygous type, which included all 9 varieties determined by previous genome resequencing and 8 local pear varieties newly collected from South China, and showed low chilling requirement in production, which was suitable for southern cultivation conditions and only required low temperature accumulation to break dormancy and complete flowering and fruiting; while the northern ‘Dangshan Su’ and ‘Hongzaosu’ pear showed CC type, which was consistent with the previous resequencing results, and showed high chilling requirement for dormancy release in production, which was not suitable for high temperature climate conditions.

[0080] 3. Sequencing the PCR product by Sanger method to verify the accuracy of KASP typing

[0081] The PCR products of 3 CA samples (‘Yeshen pear’, ‘Chihua pear’, and ‘Taiwan Chihua’) and 1 CC sample (‘Hongzaosu’) were sequenced by Sanger method, as shown in Figure 3 the sequencing peak chart of the 3 CA heterozygous samples showed double peaks at the corresponding SNP site, corresponding to A / C two amplification products, while the corresponding SNP site of the 1 CC sample showed a single peak, corresponding to C amplification product. These results showed that the KASP typing results were consistent with the Sanger sequencing results, verifying the accuracy of KASP marker typing.

[0082] In summary, the KASP marker detection kit for the single nucleotide polymorphism site of the DAM1 gene was developed according to the aforementioned application, the genotype of the CA heterozygous genotype variety found by resequencing technology was successfully verified, 8 varieties with the aforementioned CA heterozygous genotype were further identified, and the CA heterozygous and CC homozygous genotypes were distinguished, the KASP technology typing results were further compared with the Sanger sequencing results, and the accuracy of the KASP marker typing developed by the application was confirmed.

Claims

1. The use of a single nucleotide polymorphism site as a detection target in the preparation of a kit for detecting chilling requirement traits of pear, characterized in that, The single nucleotide polymorphism site is a C>A polymorphism at position 216 of the nucleotide sequence shown in SEQ ID NO.

1.

2. Use according to claim 1, wherein The kit comprises specific primers for detecting the single nucleotide polymorphism at position 216 of the nucleotide sequence shown in SEQ ID NO.

1.

3. Use according to claim 2, wherein the compound is ###0002### The specific primers are two pairs, wherein the nucleotide sequence of the upstream primer of primer pair I is shown in SEQ ID NO. 2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 4; the nucleotide sequence of the upstream primer of primer pair II is shown in SEQ ID NO. 3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.

4.

4. A KASP kit for detecting chilling requirement traits in pear, characterized in that, The KASP kit comprises a primer set for specifically detecting the single nucleotide polymorphism at position 216 of the nucleotide sequence shown in SEQ ID NO.

1.

5. The KASP kit for detecting chilling requirement traits in pear according to claim 4, wherein, The primer set comprises upstream typing primer F1, upstream typing primer F2 and downstream universal primer R, and the nucleotide sequences thereof are shown in SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 4, respectively.

6. A method for identifying low chilling requirement pear cultivars / lines, characterized in that, The method comprises the following steps: (1) extracting the genomic DNA of the pear variety to be tested as a template, and performing PCR reaction using a primer set for specifically detecting the single nucleotide polymorphism at position 216 of the nucleotide sequence shown in SEQ ID NO. 1; (2) determining the cold requirement trait of the sample to be tested according to the genotype of the product: if the genotype of the product is CA, the sample to be tested is determined to be a low cold requirement variety / strain; if the genotype of the product is CC, the sample to be tested is determined to be a high cold requirement variety / strain.

7. The method of identifying low chill pear cultivars / lines of claim 6 wherein, In step (1), the primer set with the nucleotide sequences shown in SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 4 is used for KASP reaction detection; in step (2), the reaction product is subjected to fluorescence data reading, and each sample is genotyped according to the relative intensity of different wavelengths of fluorescence: if the fluorescence is orange yellow, the genotype is CC; if the fluorescence is green, the genotype is CA.

8. The method of identifying low chill pear varieties / lines of claim 7, wherein, The composition of the KASP reaction system comprises, in a total volume of 10 μL, 1×KASP premix, a final concentration of 0.1 μM of upstream typing primer F1, a final concentration of 0.1 μM of upstream typing primer F2, a final concentration of 0.2 μM of downstream universal primer R, and 10-50 ng of genomic DNA template.

9. The method of identifying low chill pear varieties / lines of claim 7, wherein, The KASP reaction conditions are: 95℃ pre-denaturation for 10 min, 1 cycle; 95℃ denaturation for 15 s, 61-55℃ annealing and extension for 60 s, 10 cycles, with the temperature of annealing and extension decreasing by 0.6℃ for each cycle; 95℃ denaturation for 15 s, 55℃ annealing and extension for 60 s, 28-35 cycles.

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