KASP molecular marker for predicting resistance to peach gummosis and application thereof

Abstract

The application discloses a KASP molecular marker for predicting resistance of peach tree to gummosis and application thereof, and relates to the field of molecular biology. Four KASP molecular markers related to the resistance of peach to gummosis and primer combinations thereof are developed for the first time, and the KASP molecular markers are verified and evaluated in combination with phenotype data, and 510 peach varieties are typed by using the KASP molecular markers and the primer combinations, so that the resistance identification efficiency of peach germplasm is improved, and theoretical and technical support is provided for molecular marker assisted breeding.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to a KASP molecular marker for predicting resistance to peach gummosis and its application. Background Technology

[0002] Peach (Prunus persica (L.) Batsch), belonging to the genus Prunus in the family Rosaceae, is one of my country's important economic fruit trees, currently cultivated throughout the country. Peach gummosis is a common disease in peach cultivation, prevalent in hot and humid climates. The main symptom is the appearance of a gum-like substance on the tree trunk; the more severe the disease, the larger the area covered by the gum. Gummosis weakens the tree, reduces peach yield, and causes significant economic losses. Effective control of gummosis is a major problem in peach production and cultivation. Currently, production practices largely leave gummosis unchecked, with limited control relying mainly on chemical or biological methods. Developing highly resistant germplasm resources and cultivating new resistant varieties are fundamental measures to reduce peach gummosis.

[0003] Molecular marker-assisted breeding utilizes molecular markers closely linked to target traits to screen for superior genotypes, thereby improving breeding efficiency and accelerating the breeding process. Currently, a series of molecular markers related to important peach traits have been developed, including quality traits such as peel (hairy / hairless), fruit shape (flat / round), flesh texture (firm / soft), and flesh color (red / white / yellow) (Yang Yingjun, Zhang Kaichun, Li Rongqi, et al. RAPD molecular markers for hairy / hairless and white / yellow flesh traits in peach fruit [J]. North China Agricultural Journal, 2000, 15(003): 6-9.), and aphid resistance. Disease resistance traits such as resistance to root-knot nematodes (Meng Junren, Zeng Wenfang, Deng Li, et al. Development and application of KASP molecular markers for several important traits of peach [J]. Chinese Agricultural Science, 2021.), and morphological traits such as flower shape (bell-shaped / rose-shaped) (Ji Shuangqiu, Wang Lirong, Li Yong, et al. Genotyping of peach flower shape (bell-shaped / rose-shaped), development and utilization of molecular markers [J]. Journal of Fruit Science, 2023, 40(3): 422-431.). However, research on molecular markers related to gummosis resistance is rarely reported.

[0004] With the completion of peach whole genome sequencing, genetic linkage mapping technology and genome-wide association analysis have become important means to discover target genes and develop molecular markers. Daniel (MANCERO-CASTILLO D, BECKMAN TG, HARMON PF, et al. A major locus for resistance to Botryosphaeria dothidea in Prunus. Tree Genetics & Genomes, 2018, 14(2): 26. 2018) discovered a dominant resistance gene from almond by constructing a hybrid population and genetic mapping analysis, and located it in the 6th-8th chimeric linkage group region of peach. Gan Kexin (2021) obtained a series of peach gummosis-related SNP sites through resequencing BSA association analysis. Li (2022) used resequencing GWAS association analysis combined with RNA-seq to identify 5 significant SNP sites and 4 candidate genes related to gummosis resistance. With the development of sequencing technology, competitive allele-specific PCR (KASP) markers have been applied to molecular-assisted breeding due to their high throughput and low cost. KASP utilizes the specificity of competing alleles to genotype single nucleotide polymorphisms (SNPs) and insertions / deletions (indels) at specific loci. Developing KASP markers associated with peach gummosis using these resistance-related SNP loci allows for rapid and effective screening of highly resistant varieties, providing a theoretical basis for marker-assisted breeding and the cultivation of new disease-resistant peach varieties. Summary of the Invention

[0005] The purpose of this invention is to provide a KASP molecular marker for predicting resistance to gummosis in peach trees. This KASP molecular marker is located on linkage groups 1, 2, 4, and 6 of peach, and is significantly correlated with the incidence of gummosis in peach trees. It exhibits good polymorphism. Using this KASP molecular marker and its primer combination to detect gummosis resistance in different peach varieties or lines has high accuracy, which is beneficial to improving the breeding efficiency of peach trees and accelerating the breeding of new gummosis-resistant peach varieties in high-temperature and high-humidity areas.

[0006] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0007] A KASP molecular marker for predicting resistance to peach gummosis, wherein the KASP molecular marker is at least one of the following:

[0008] (1)Pp01_37618213; (2)Pp02_13386543; (3)Pp04_25252602; (4)Pp06_27085141;

[0009] The Pp01_37618213 site is located at position 37618213 on chromosome 1, and the base at this site is either T or C; the alleles T and C that locate Pp01_37618213 are the 101st base of the sequence shown in SEQ ID NO.13;

[0010] The Pp02_13386543 site is located at position 13386543 on chromosome 2, and the base at this site is either T or C; the alleles T and C that locate Pp02_13386543 are the 101st base of the sequence shown in SEQ ID NO.14;

[0011] The Pp04_25252602 locus is located at position 25252602 on chromosome 4, and the base at this locus is either T or C; the alleles T and C that locate Pp04_25252602 are the 101st base of the sequence shown in SEQ ID NO.15;

[0012] The Pp06_27085141 site is located at position 27085141 on chromosome 6. The bases at this site are either T or C. The alleles T and C that locate Pp06_27085141 are the 101st bases of the sequence shown in SEQ ID NO.16.

[0013] This invention also provides the application of the KASP molecular marker for predicting resistance to peach gummosis in the breeding of peach varieties or lines.

[0014] This invention also provides a KASP molecular marker primer pair for predicting resistance to peach gummosis, specifically a primer pair targeting the Pp01_37618213 site:

[0015] The forward FAM primer sequence is shown in SEQ ID No. 1, the forward HEX primer sequence is shown in SEQ ID No. 2, and the universal reverse primer sequence is shown in SEQ ID No. 3;

[0016] Primer pair targeting the Pp02_13386543 site:

[0017] The forward FAM primer sequence is shown in SEQ ID No. 4, the forward HEX primer sequence is shown in SEQ ID No. 5, and the universal reverse primer sequence is shown in SEQ ID No. 6;

[0018] Primer pair targeting the Pp04_25252602 site:

[0019] The forward FAM primer sequence is shown in SEQ ID No. 7, the forward HEX primer sequence is shown in SEQ ID No. 8, and the universal reverse primer sequence is shown in SEQ ID No. 9;

[0020] Primer pair targeting the Pp06_27085141 site:

[0021] The forward FAM primer sequence is shown in SEQ ID No. 10, the forward HEX primer sequence is shown in SEQ ID No. 11, and the universal reverse primer sequence is shown in SEQ ID No. 12;

[0022] The forward FAM primer has a fluorescent label FAM attached to its 5' end, and the forward HEX primer has a fluorescent label HEX attached to its 5' end.

[0023] The present invention also provides a kit for predicting resistance to peach gummosis, comprising the aforementioned KASP molecular marker primer combination.

[0024] This invention also provides a method for predicting resistance to gummosis in peach trees, comprising the following steps:

[0025] (1) Extract genomic DNA from the peach tissue to be tested;

[0026] (2) Using the genomic DNA as a template, the KASP molecular marker primer combination was used to perform PCR amplification on the Pp01_37618213 site, Pp02_13386543 site, Pp04_25252602 site, and Pp06_27085141 site to obtain PCR amplification products.

[0027] (3) Genotyping of the PCR amplification products to obtain the genotypes of Pp01_37618213, Pp02_13386543, Pp04_25252602 and Pp06_27085141 sites respectively.

[0028] If the genotype at the Pp01_37618213 locus in the genome of the peach variety being tested is TT, it is a highly resistant peach variety; if the genotype at the Pp01_37618213 locus is CT or TT, it is a low-resistant peach variety. If the genotype at the Pp02_13386543 locus in the genome of the peach variety being tested is CC, it is a highly resistant peach variety; if the genotype at the Pp02_133865433 locus is CT or TT, it is a low-resistant peach variety. A peach variety with the genotype TT at the Pp04_25252602 locus is highly resistant, while one with the genotype CC at the Pp04_25252602 locus is less resistant. Similarly, a peach variety with the genotype CC at the Pp06_27085141 locus is highly resistant, while one with the genotype TT at the Pp06_27085141 locus is less resistant. The incidence of gummosis in highly resistant peach varieties is significantly lower than in less resistant varieties.

[0029] Furthermore, in step (3), when the genotype CT at Pp01_37618213 and the genotype TT at Pp04_25252602 form a gene combination, it is a low-resistant peach variety; when the genotype CC at Pp04_25252602 and the genotype TT at Pp06_27085141 form a gene combination, it is a high-resistant peach variety.

[0030] Using the obtained degraded and enhanced genotypes, the phenotypic effects of different genotype combinations on gummosis resistance were analyzed. The optimal genotype combination for evaluating highly resistant materials, Pp04_25252602+Pp06_27085141-C / C+T / T, was selected, with a selection rate of 82.76%. The optimal genotype combination for evaluating low-resistance materials, Pp01_37618213+Pp04_25252602-C / T+T / T, was selected, with a selection rate of 92.86%.

[0031] Specifically, in step (2), the pre-denaturation is carried out at 94℃ for 15 min; denaturation is carried out at 94℃ for 20 s, and extension is carried out at 61℃ for 60 s. The temperature is reduced by 0.6℃ in each extension cycle, and the cycle is repeated 10 times; denaturation is carried out at 94℃ for 20 s, and extension is carried out at 55℃ for 60 s, and the cycle is repeated 26 times.

[0032] If the signal value is still low after 26 cycles, and the clusters are not dispersed but show a clustering trend, choose to add a cycle reaction before fluorescence reading. Cycling reaction: denaturation at 94℃ for 20s, extension at 57℃ for 60s, 3 cycles.

[0033] The beneficial effects of this invention are as follows: This invention is the first to develop four KASP molecular markers and their primer combinations associated with peach gummosis resistance, which are then verified and evaluated in combination with phenotypic data. Furthermore, the KASP markers and their primer combinations are used to genotype 510 peach varieties, thereby improving the efficiency of peach germplasm resistance identification and providing theoretical and technical support for molecular marker-assisted breeding. Attached Figure Description

[0034] Figure 1 This section presents whole-genome variation data and variation maps; where A represents statistical data obtained from whole-genome resequencing; and B represents the CIRCOS whole-genome variation map, with each ring from the outside to the inside representing chromosomes, GC distribution, INDEL density, and SNP density distribution, respectively.

[0035] Figure 2 The results of BSA association analysis for nectarines (top) and common peaches (bottom);

[0036] Figure 3 The distribution of monomeric blocks is associated with significant sites;

[0037] Figure 4 The typing results for the four KASP tags;

[0038] Figure 5 This is the direct sequencing result for the Pp06_27085141 locus;

[0039] Figure 6 To compare the differences in the severity of gummosis among different genotypes; where ns P>0.05, *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 (Tukey's test). Detailed Implementation

[0040] Example 1: Obtaining the resistance-associated loci and KASP markers for peach gummosis

[0041] 1. Whole genome resequencing of peach varieties

[0042] Genomic DNA extraction: Ten highly resistant materials (gummosis grade less than 1, according to the Hubei Academy of Agricultural Sciences. Grading and Integrated Control Technical Regulations for Peach Gummosis: DB42 / T 1183-2016) and ten highly resistant materials (gummosis grade greater than 3) were selected from common peach varieties. Similarly, ten highly resistant materials (gummosis grade less than 2) and ten highly resistant materials (gummosis grade greater than 5) were selected from nectarine varieties. Genomic DNA pools for common peach and nectarine groups were constructed separately. Peach genome extraction was performed using the CTAB extraction method, with the specific steps as follows:

[0043] (1) Take 1-2g of leaf, add 2 clean small steel balls to a 2mL PE tube, and grind for 60s at a frequency of 60Hz using a fully automatic grinder;

[0044] (2) Add 400 μL of CTAB (2% CTAB, 20 mM EDTA, 0.1 M Tris, 1.4 M NaCl, pH = 8.0) and 10 μL of β-mercaptoethanol, and incubate in a water bath at 65°C for 1 h, shaking the mixture up and down every 15 min during the incubation period.

[0045] (3) Add 400 μL of chloroform:isoamyl alcohol (24:1), mix well, centrifuge at 10000 rpm for 10 min, transfer the supernatant to a new centrifuge tube, add an equal volume of chloroform:isoamyl alcohol (24:1), mix well, and centrifuge at 10000 rpm for 10 min.

[0046] (4) Take out the supernatant, add 2 / 3 volume of isopropanol pre-cooled at -20℃, mix well, and let stand overnight at -20℃.

[0047] (5) Centrifuge at 10000 rpm for 15 min. A white precipitate will be visible. Discard the supernatant, add 500 μL of 75% ethanol, shake and wash the precipitate, and centrifuge at 8000 rpm for 4 min.

[0048] (6) Discard the supernatant, add 500 μL of 100% ethanol, shake to wash the precipitate, and centrifuge at 8000 rpm for 4 min;

[0049] (7) Remove excess supernatant, place in a fume hood and blow dry for 1 hour until completely dry, then add 50-100 μL of TE solution to dissolve the precipitate.

[0050] Whole-genome resequencing: The obtained peach genomic DNA was adjusted to a concentration of 200 μg / ml, and 100 μL was used for sequencing. The sequencing platform system used was Illumina Novaseq 6000, PE150, with a sequencing depth of 10X and a sequencing data volume of 3G. Low-quality sequences were filtered using FastP software. The reference genome used was Prunus persica Whol e GenomeAssembly v2.0 & Annotation v2.1 (v2.0.a1) (GDR: https: / / www.rosaceae.org / species / prunus_persica / genome_v2.0.a1). Clean data was compared with the reference genome using BWA software to detect variant sites, obtaining 3,000,342 SNPs (e.g., ...). Figure 1 (As shown).

[0051] 2. Association analysis of peach BSA resequencing

[0052] BSA association analysis was used to screen for loci associated with gummosis, and ED association analysis was used to calculate the ED value of SNP loci. The calculation formula is as follows.

[0053]

[0054] Here, mut and wt represent pools with two different traits, and A, C, G, and T represent the proportion of sequencing reads for each mutant at the marker site. Based on the obtained SNP locus sets and genotype depth information between pools, the mutation frequency difference, i.e., the ED value, is calculated. A threshold of 1.00 is set; loci exceeding this threshold are considered significantly associated SNPs. 83 associated loci were detected in the nectarine group, and 50 associated loci were detected in the regular peach group (e.g., ...). Figure 2 (As shown).

[0055] 3. Single-unit block analysis

[0056] The LDBlockShow software was used to perform haplotype block (LD block) analysis on the 133 associated loci obtained in step 2. Default parameters (MAF 0.05, Miss 0.25) were used, and a sliding window method was employed for detection. The search unit was 1 Mb. The output data of the genome-wide variant locus block detection were analyzed, and 70 significant loci with small-scale associated haplotype blocks (0.004 kb-11.954 kb) were identified. This resulted in 51 blocks and 692 associated loci (e.g., ...). Figure 3 (As shown).

[0057] 4. Development of KASP tags

[0058] The associated loci obtained in steps 2 and 3 were screened to obtain 56 candidate loci. The screening principles are as follows: 1. The number of other polymorphic loci within 50 bp upstream and downstream of the locus should be less than 2; 2. If there are multiple loci in the same haplotype block, the locus with the highest ED value in the same haplotype block is selected; 3. Combining the gelling disease phenotypic data and the previous resequencing data, the loci with the larger genotypic difference between high-resistance and low-resistance materials are selected.

[0059] Utilizing Kraken of the UK Government Chemist Laboratory (LGC) TMThe software designed KASP primers for 28 candidate sites, with the following design principles: (1) the GC content of the primer sequence is between 30% and 60%; (2) the length of the primer sequence is between 18 and 28 bp; (3) the length of the amplified product sequence is between 80 and 120 bp; (4) the primers have good specificity and can amplify the target fragment only in the peach genome. Each group includes two specific forward primers and one universal reverse primer. The 5' ends of the two forward primers are connected to different fluorescent tag adapter sequences (FAM-GAAGGTGACCAAGTTCATGCT, HEX-GAAGGTCGGAGTCAACGGATT), while the 3' end bases are the variation sites.

[0060] Based on the above sites and using Kraken TM A set of KASP primers was designed using software. The molecular markers included Ppc1_37618213 (sequence shown in SEQ ID NO.13), Pp02_13386543 (sequence shown in SEQ ID NO.14), Pp04_25252602 (sequence shown in SEQ ID NO.15), and Pp06_27085141 (sequence shown in SEQ ID NO.16). The specific sequence information of the primers is shown in Table 1 below.

[0061] Table 1

[0062]

[0063] Example 2: Validation of KASP markers associated with peach gummosis resistance

[0064] 1. PCR amplification

[0065] Eighty-two varieties with known resistance to gummosis were selected, and the developed KASP marker was detected using an IntelliQube instrument in the “SNP genotyping inline mode” (single membrane). The total PCR reaction volume was 1.6 μL, including 0.8 μL DNA template (diluted to 20 ng / μL), 0.8 μL 2×KASP Master mix, and 0.022 μL primer mixture (forward FAM primer:forward HEX primer:reverse universal primer:ddH2O = 6:6:15:23). The PCR cycle reaction consisted of three steps: (1) 94℃ pre-denaturation for 15 min; (2) 94℃ denaturation for 20 s, 61℃ extension for 60 s (each cycle decreased by 0.6℃), 10 cycles; (3) 94℃ denaturation for 20 s, 55℃ extension for 60 s, 26 cycles. If the signal value remains low after 26 cycles, and the clustering is not dispersed but shows a clustering trend, then an additional cycle reaction should be performed before fluorescence reading. The cycle reaction is: denaturation at 94℃ for 20 s, extension at 57℃ for 60 s, repeated 3 times.

[0066] After the PCR reaction, fluorescence data were read and analyzed using an IntelliQube machine. Four markers (Pp01_37618213, Pp02_13386543, Pp04_25252602, Pp06_27085141) yielded good-quality genotyping results on most samples, exhibiting three genotyping results: red dots near the X-axis represented homozygous genotypes carrying FAM fluorescence signals; blue dots near the Y-axis represented homozygous genotypes carrying HEX fluorescence signals; and purple dots between the two genotypes represented heterozygous genotypes carrying both FAM and HEX fluorescence signals (e.g., ...). Figure 4 (As shown).

[0067] 2. Verification using routine Sanger sequencing

[0068] Conventional primers were designed to amplify 500bp sequences upstream and downstream of the variant site. Snapgene was used to view the forward and reverse sequencing results of the amplified products to determine the genotyping status of the variant site (e.g., Figure 5 As shown in the figure, the consistency between KASP marker genotyping results and Sanger sequencing genotyping results was compared. The results showed that the KASP genotyping results of 7 markers were completely consistent with the Sanger sequencing genotyping results (Pp01_37618213, Pp06_27085141), and the genotyping results of 2 markers had a consistency of 93.75% with the Sanger sequencing results (Pp02_13386543, Pp04_25252602).

[0069] Example 3: Evaluation of the selection effect of KASP markers associated with peach gummosis resistance

[0070] 1. Correlation analysis and comparison of differences in gummosis severity

[0071] Using the above four pairs of KASP markers, 112 peach varieties with known resistance to gummosis were tested. Combining the genotyping results with gummosis phenotypic data, a unit point F-test was performed using the "Single-Locus F-test" procedure in Powermarker V3.25. It was found that all eight pairs of KASP markers were significantly associated with gummosis resistance (P < 0.01) (Table 2).

[0072] Table 2 Correlation analysis between KASP markers and phenotypes

[0073] mark F-statistic p-value Pp01_37618213 22.60 6.17E-09 Pp06_27085141 21.55 1.30E-08 Pp04_25252602 28.85 1.03E-10 Pp02_13386543 14.20 3.35E-06

[0074] 2. Evaluation of the selection effect at different sites

[0075] The phenotypic effect value was calculated using the formula (Ai = ∑Xij / ni - ∑Nk / nk), where Ai represents the phenotypic effect value of the i-th locus, Xij is the measured value of the gummosis phenotype of the j-th material carrying the i-th locus, ni is the number of materials with the i-th locus, and ∑Nk / nk represents the average of the measured phenotypic values ​​of all materials. When Ai < 0, the locus was considered ineffective (indicated by a negative sign "-"), and when Ai > 0, the locus was considered effective (indicated by a plus sign "+"). For ineffective loci, the smaller the effect value, the more significant the ineffectiveness and the stronger the gummosis resistance; for effective loci, the larger the effect value, the more significant the effectiveness and the weaker the gummosis resistance. The dominant disease resistance variant sites for each marker are: Pp01_37618213-T, Pp02_13386543-C, Pp04_25252602-C, and Pp06_27085141-T; the dominant disease susceptibility variant sites for each marker are: Pp01_37618213-C, Pp02_13386543-T, Pp04_25252602-T, and Pp06_27085141-C (as shown in Table 3).

[0076] Table 3. Selection effect analysis of variant sites

[0077]

[0078]

[0079] 3. Evaluation of the selection effect of different genotypes

[0080] The significance of phenotypic differences among materials with different genotypes was analyzed using the multiple comparisons function of Graphpad 9.0 (e.g., Figure 6 As shown in the figure, the gummosis severity of materials carrying different loci in each marker group showed highly significant differences (P < 0.01). The selection rate of each marker genotype for highly resistant (or low-resistant) materials was calculated using the formula: Selection rate of a marker for a certain phenotype = Number of materials carrying the marker genotype and exhibiting the certain phenotype / Total number of materials carrying the marker genotype. The results are shown in Table 4. Four degraded genotypes were obtained: Ppμ1_37618213-T / T, Pp02_13386543-C / C, Pp04_25252602-C / C, and Pp06_27085141-T / T. Four enhanced genotypes were also obtained: Pp01_37618213-C / T, Pp02_13386543-T / T, Pp04_25252602-T / T, and Pp06_27085141-C / C.

[0081] Table 4. Analysis of the selection effect of different genotypes

[0082]

[0083] 4. Evaluation of the selection effect of genotype combinations

[0084] Using the degraded and enhanced genotypes obtained in step 3, the phenotypic effects of different genotype combinations on gummosis resistance were analyzed. The optimal genotype combination for evaluating highly resistant materials, Pp04_25252602+Pp06_27085141-C / C+T / T, was selected, with a selection rate of 82.76%. The optimal genotype combination for evaluating low-resistant materials, Pp01_37618213+Pp04_25252602-C / T+T / T, was also selected, with a selection rate of 92.86% (as shown in Tables 5 and 6).

[0085] Table 5. Selection effect analysis of genotype combinations with reduced efficacy.

[0086]

[0087]

[0088] Table 6. Analysis of the selection effect of synergistic genotype combinations.

[0089]

[0090] Example 4: Application of KASP markers in peach gummosis resistance association

[0091] Genotyping of 510 peach varieties was performed using the four KASP markers associated with peach gummosis resistance (as shown in Table 7). The superiority-enhancing genotype combination and the superiority-reducing genotype combination obtained in Example 3 were used to evaluate the 510 peach varieties.

[0092] Table 7: KASP typing results of gummosis resistance in 510 peach varieties

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100] Note: ". / ." indicates no fluorescence signal was detected. Among them, Pp01 represents Pp01_37618213, Pp02 represents Pp02_13386543, Pp04 represents Pp04_25252602, and Pp06 represents Pp06_27085141.

[0101] Using the combination Pp04_25252602+Pp06_27085141-C / C+T / T, 120 potentially highly resistant varieties were initially screened, including 24 known highly resistant varieties to gummosis. Using the combination Pp01_37618213+Pp04_25252602-C / T+T / T, 31 potentially low-resistant varieties were initially screened, including 14 known low-resistant varieties to gummosis.

Claims

1. The application of KASP molecular markers for predicting resistance to peach gummosis in peach variety or strain breeding, wherein the KASP molecular marker is at least one of the following: (1) Pp01_37618213; (2) Pp02_13386543; (3) Pp04_25252602; (4) Pp06_27085141; in, Pp01_37618213 is located on chromosome 1 of peach. The nucleotide sequence of Pp01_37618213 is shown in SEQ ID NO.13, with the 101st base being either T or C. Pp02_13386543 is located on chromosome 2 of peach. The nucleotide sequence of Pp02_13386543 is shown in SEQ ID NO.14, with the 101st base being either T or C. Pp04_25252602 is located on chromosome 4 of peach. The nucleotide sequence of Pp04_25252602 is shown in SEQ ID NO.15, with the 101st base being either T or C. Pp06_27085141 is located on chromosome 6 of peach. The nucleotide sequence of Pp06_27085141 is shown in SEQ ID NO.16, with the 101st base being either T or C.

2. A KASP molecular marker primer combination for predicting resistance to peach gummosis, characterized in that, The KASP molecular marker primer combination includes: primer pairs targeting the Pp01_37618213 site: The forward FAM primer sequence is shown in SEQ ID No. 1, the forward HEX primer sequence is shown in SEQ ID No. 2, and the universal reverse primer sequence is shown in SEQ ID No. 3; Primer pair targeting the Pp02_13386543 site: The forward FAM primer sequence is shown in SEQ ID No. 4, the forward HEX primer sequence is shown in SEQ ID No. 5, and the universal reverse primer sequence is shown in SEQ ID No. 6; Primer pair targeting the Pp04_25252602 site: The forward FAM primer sequence is shown in SEQ ID No. 7, the forward HEX primer sequence is shown in SEQ ID No. 8, and the universal reverse primer sequence is shown in SEQ ID No. 9; Primer pair targeting the Pp06_27085141 site: The forward FAM primer sequence is shown in SEQ ID No. 10, the forward HEX primer sequence is shown in SEQ ID No. 11, and the universal reverse primer sequence is shown in SEQ ID No. 12; The forward FAM primer has a fluorescent label FAM attached to its 5' end, and the forward HEX primer has a fluorescent label HEX attached to its 5' end.

3. A kit for predicting resistance to gummosis in peach trees, characterized in that, Includes the KASP molecular marker primer combination as described in claim 2.

4. A method for predicting resistance to gummosis in peach trees, characterized in that, Includes the following steps: (1) Extract genomic DNA from the peach tissue to be tested; (2) Using the genomic DNA as a template, PCR amplification was performed on the Pp01_37618213 site, Pp02_13386543 site, Pp04_25252602 site, and Pp06_27085141 site using the KASP molecular marker primer combination as described in claim 2, to obtain PCR amplification products; (3) Genotyping of the PCR amplification products to obtain the genotypes of Pp01_37618213, Pp02_13386543, Pp04_25252602 and Pp06_27085141 sites respectively; If the genotype at the Pp01_37618213 locus of the peach variety being tested is TT, it is a highly resistant peach variety; if the genotype at the Pp01_37618213 locus is CT or TT, it is a low-resistant peach variety. If the genotype at the Pp02_13386543 locus in the genome of the peach variety being tested is CC, it is a highly resistant peach variety; if the genotype at the Pp02_133865433 locus is CT or TT, it is a low-resistant peach variety. If the genotype at the Pp04_25252602 locus in the genome of the peach variety being tested is TT, it is a highly resistant peach variety; if the genotype at the Pp04_25252602 locus is CC, it is a low-resistant peach variety. If the genotype at the Pp06_27085141 locus in the genome of the peach variety being tested is CC, it is a highly resistant peach variety; if the genotype at the Pp06_27085141 locus is TT, it is a low-resistant peach variety. Among them, the incidence of gummosis in peach trees of highly resistant peach varieties was significantly lower than that in low-resistant peach varieties.

5. The method for predicting peach tree gummosis resistance as described in claim 4, characterized in that, In step (3), when the genotype CT at the Pp01_37618213 locus and the genotype TT at the Pp04_25252602 locus together form a gene combination, it is a low-resistance peach variety. When the genotype CC at the Pp04_25252602 locus and the genotype TT at the Pp06_27085141 locus combine to form a gene combination, it results in a highly resistant peach variety.

6. The method for predicting peach tree gummosis resistance as described in claim 4, characterized in that, In step (2), the pre-denaturation is carried out at 94℃ for 15 min; denaturation is carried out at 94℃ for 20 s, followed by extension at 61℃ for 60 s, with the temperature decreasing by 0.6℃ in each extension cycle, for a total of 10 cycles; denaturation is carried out at 94℃ for 20 s, followed by extension at 55℃ for 60 s, for a total of 26 cycles.

7. The method for predicting peach tree gummosis resistance as described in claim 6, characterized in that, If the fluorescence signal value cannot be read after 26 cycles, select to add a cycle reaction before fluorescence reading; the cycle reaction is: denaturation at 94℃ for 20 s, extension at 57℃ for 60 s, for 3 cycles.

Images