KASP Technology-Based SNP Molecular Markers for Fruit Mulberry against Sclerotinia Disease and Their Applications

Abstract

The present application discloses a SNP molecular marker for fruit mulberry resistant to Sclerotinia sclerotiorum developed based on the KASP technology and its application. The SNP molecular marker comprises a nucleotide sequence formed by a single nucleotide mutation of A>T at the 18,208,100th base of chromosome No. 4 of the Morus alba genome. Based on the KASP technology and combined with this SNP molecular marker, the present application provides a method for identifying fruit mulberry varieties resistant to Sclerotinia sclerotiorum and / or anthracnose, which can be accurately used for genotyping of resistant and susceptible genotypes of fruit mulberry, greatly shortening the breeding cycle of disease-resistant mulberry varieties. This method is convenient, fast, has a high detection throughput, low cost and is not affected by the environment.

Description

Technical Field

[0001] The present application relates to the technical field of fruit mulberry resource identification, and in particular to fruit mulberry anti-sclerotinia sclerotiorum SNP molecular markers developed based on KASP technology and their application. Background Art

[0002] Mulberry is an important economic tree species, with a cultivation history of more than 5,000 years in my country. Mulberry fruit is not only sweet and juicy, unique in taste and delicious, but also rich in nutrition. It contains rich sugar, acid and multiple vitamins, amino acids, trace elements and other minerals, flavonoids, etc., especially selenium content is the highest among all fruits. It has pharmacological effects such as promoting the growth of hematopoietic cells, lowering blood sugar and blood lipids, and was listed as "both food and medicine" by the former Ministry of Health. In addition to direct consumption, mulberry has been developed into fruit juice drinks, mulberry wine, mulberry jam, mulberry paste and anthocyanins, with broad market prospects. my country has collected and preserved more than 200 fruit mulberry resources, most of which belong to Guangdong mulberry and white mulberry. So far, fruit mulberry varieties such as Yuezhen Dashi, Suiguo No. 2, Hongguo No. 1, Jialing No. 30, and Jialing No. 40 have been bred, but mulberry sclerotinia has become a bottleneck problem restricting the development of the fruit mulberry industry. Mulberry sclerotinia is a severe disease with a rapid onset. In some places, thousands of acres of mulberry orchards have been completely wiped out due to the disease, causing devastating damage to the mulberry industry. Therefore, cultivating new mulberry varieties resistant to sclerotinia is currently the most effective and economical means to solve the problem of industrial development.

[0003] The breeding of mulberry varieties is mainly carried out through three ways: selective breeding, hybrid breeding and mutagenesis breeding. Selective breeding can only select excellent individuals from local varieties or natural variations, and cannot innovate purposefully. Mutagenesis breeding currently still has problems such as the inability to control induced mutations, the low rate of favorable mutations, and the need for a large number of selections. Hybrid breeding is still the most widely used and effective way of mulberry breeding. Due to the long cycle from sowing to selecting disease-resistant individual plants for fruit mulberry hybrid offspring, all individual plant traits need to be identified during artificial selection, which is time-consuming and labor-intensive, and environmental conditions affect the accuracy of disease resistance evaluation. Therefore, it is of great significance to explore disease-resistant genes and develop molecular marker-assisted selection (MAS) technology to evaluate hybrid individual plants.

[0004] According to the level of technological development, DNA molecular markers can be divided into three generations. The first-generation molecular markers are RFLP (Restriction Fragment Length Polymorphism) markers based on Southern hybridization technology. The first-generation molecular marker technology is cumbersome to operate, has a long detection period, high cost, and low success rate. The second-generation molecular markers are mainly RAPD (Random Amplified Polymorphic DNA), AFLP (Amplified Fragment Length Polymorphism), SSR (Simple Sequence Repeat), SCAR (Sequence Characterized Amplified Region), and ISSR (Inter-Simple Sequence Repeat) markers developed based on the core of PCR technology. The second-generation molecular marker technology involves relatively cumbersome traditional gel electrophoresis steps, poor experimental repeatability, and is prone to generating false positive bands. To obtain good repeatability, strict experimental conditions must be controlled, and it has low throughput. The third-generation molecular markers are mainly SNPs (Single Nucleotide Polymorphisms), which have the advantages of high throughput, high accuracy, and easy data integration, and have become the main technical methods for molecular-assisted breeding and variety identification.

[0005] At present, there is no report on the mining of the resistance genes against Sclerotinia sclerotiorum in mulberry fruits and the development of closely linked molecular markers at home and abroad, and the resistance mechanism is still not very clear. Summary of the Invention

[0006] Competitive allele specific PCR (KASP) technology is a fluorescence detection genotyping technology. This technology is based on the specific matching of the base at the end of the primer to genotype SNPs and detect insertions or deletions (InDels) in the gene sequence. This detection technology can use 2 fluorescence probes, 2 universal quenching probes, and multiple locus-specific probes to detect multiple SNP loci. The technical operation is simple, the analysis is stable and accurate, and the cost is relatively low, making it easy to achieve high throughput and automation.

[0007] Aiming at the problems of the existing technology, the purpose of this application is to provide a SNP molecular marker for mulberry fruits resistant to Sclerotinia sclerotiorum, and use this marker for the breeding of mulberry fruit varieties resistant to Sclerotinia sclerotiorum.

[0008] In the previous research of the present applicant, the transcriptome sequencing data of high-resistant and high-susceptible mulberry varieties in response to the infection of Sclerotinia sclerotiorum and the transcriptome sequencing data of high-resistant and high-susceptible mulberry varieties in response to the infection of Colletotrichum gloeosporioides were analyzed, and it was found that some genes responded to both diseases. Through functional verification of the genes responding to both diseases by virus-induced gene silencing (VIGS) and transient overexpression, it was found that these genes have the functions of resisting Sclerotinia sclerotiorum and Colletotrichum gloeosporioides. On this basis, in the embodiments of the present application, the KASP molecular marker technology was used to identify the gene loci resistant to Sclerotinia sclerotiorum in mulberry resources or breeding combinations, realizing molecular design breeding for resisting Sclerotinia sclerotiorum, which is beneficial to the transfer and aggregation of specific genes, accelerating the breeding progress of resistant varieties, avoiding blindness in traditional breeding, saving breeding costs, and improving the accuracy and efficiency of breeding selection, and has important application significance.

[0009] Based on this, the present application provides the following technical solutions:

[0010] In the first aspect, the embodiments of the present application provide an SNP molecular marker related to mulberry resistance to Sclerotinia sclerotiorum, and the SNP molecular marker comprises a nucleotide sequence formed by a single nucleotide mutation of A>T at the 18,208,100th base of chromosome 4 of the Morus alba genome.

[0011] Preferably, the SNP molecular marker comprises the nucleotide sequence shown in SEQ ID NO: 1, and in the nucleotide sequence shown in SEQ ID NO: 1, N is A or T.

[0012] More preferably, the nucleotide sequence of the SNP molecular marker is as shown in SEQ ID NO: 1, wherein N is A or T.

[0013] In the second aspect, the embodiments of the present application provide a primer set for amplifying the foregoing SNP molecular marker, and the primer set is:

[0014] SNP6-F1: The nucleotide sequence is as shown in SEQ ID NO: 12;

[0015] SNP6-F2: The nucleotide sequence is as shown in SEQ ID NO: 13;

[0016] SNP6-R: The nucleotide sequence is as shown in SEQ ID NO: 14.

[0017] In the third aspect, the embodiments of the present application provide a nucleic acid molecule related to mulberry resistance to Sclerotinia sclerotiorum and / or Colletotrichum gloeosporioides, and the nucleic acid molecule comprises the foregoing SNP molecular marker.

[0018] In the fourth aspect, the embodiments of the present application provide a kit for identifying mulberry resistance to Sclerotinia sclerotiorum or Colletotrichum gloeosporioides, which comprises the foregoing primer set.

[0019] Fifth aspect, an embodiment of the present application provides a method for identifying mulberry varieties resistant to Sclerotinia sclerotiorum and / or Colletotrichum gloeosporioides, including:

[0020] Using the DNA of the mulberry to be tested as a template, performing PCR amplification on it with the aforementioned primer set; directly distinguishing the genotype and the correlation between the genotype and the mulberry variety through fluorescence signals to identify mulberry varieties resistant to Sclerotinia sclerotiorum and / or Colletotrichum gloeosporioides.

[0021] Sixth aspect, an embodiment of the present application provides the application of the aforementioned molecular marker or the aforementioned primer set or the aforementioned kit or the aforementioned method in mulberry breeding. Description of the Drawings

[0022] Figure 1 It is a Venn diagram of the population-specific SNP distribution of resistant and susceptible mulberry varieties to Sclerotinia sclerotiorum provided by an embodiment of the present application.

[0023] Figure 2 It is the KASP genotyping detection of some samples of the primer set of the SNP6 molecular marker provided by an embodiment of the present application; where A is the detection result of 26 mulberry resources, and B is the detection result of a partial hybrid F1 population of Pearl White × Kangqing No. 10.

[0024] Figure 3 It is the verification result of the disease resistance of the KASP molecular marker provided by an embodiment of the present application; where A and B are the situations of inoculating Colletotrichum gloeosporioides 3 months after the hybrid seedlings are germinated and sown, C is the statistical result of the incidence rate after inoculating Colletotrichum gloeosporioides on the hybrid seedlings, and D is the correlation result between the incidence rate and the KASP genotyping. Detailed Embodiments

[0025] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application. The reagents not specifically described in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not specifically described in detail are all conventional experimental methods and can be learned from the prior art.

[0026] It should be noted that in the text of the present application, "ALE2" is the abbreviation of the full gene name "receptor-like serine / threonine-protein kinase ALE2", and the two represent the same gene.

[0027] The embodiments of the present application provide a mulberry antibacterial sclerotinia SNP molecular marker developed based on the KASP technology and its application. In the embodiments of the present application, 27 mulberry resources were subjected to genome resequencing, and GWAS genome-wide association analysis was performed, and 10 SNP molecular markers were designed, namely SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, SNP8, SNP9, and SNP10. SNaPshot was used for verification, and it was found that 3 SNP6, SNP7, and SNP8 molecular markers resistant to mulberry sclerotinia had an accuracy rate of more than 93%. According to the aforementioned 3 SNPs, KASP primers were designed, and it was found that only SNP6 was successfully used for genotyping. The KASP molecular marker genotyping results were consistent with the field sclerotinia survey results, indicating that it can be used for the identification of mulberry sclerotinia resistance.

[0028] In the previous research of the present application, disease-resistant genes were mined through transcriptome sequencing, and functional verification was carried out through methods such as VIGS and transient overexpression. It was found that some genes had dual functions of antibacterial sclerotinia and anthracnose. After inoculating the hybrid seedling F1 population with anthracnose bacteria, the incidence rate was correlated with the KASP results, and it was found that this SNP6 molecular marker could also be used for the identification of anthracnose resistance.

[0029] Based on this, the embodiments of the present application provide an SNP molecular marker related to mulberry antibacterial sclerotinia. The SNP molecular marker includes a nucleotide sequence formed by a single nucleotide mutation of A>T at the 18,208,100th base of chromosome 4 of the white mulberry genome.

[0030] In some embodiments, the SNP molecular marker includes the nucleotide sequence shown in SEQ ID NO: 1, wherein, in the nucleotide sequence shown in SEQ ID NO: 1, N is A or T.

[0031] In some preferred embodiments, the nucleotide sequence of the SNP molecular marker is as shown in SEQ ID NO: 1, wherein N is A or T.

[0032] Based on this, the embodiments of the present application provide a primer set for amplifying the aforementioned molecular marker. The primer set is:

[0033] SNP6-F1: The nucleotide sequence is as shown in SEQ ID NO: 12;

[0034] SNP6-F2: The nucleotide sequence is as shown in SEQ ID NO: 13;

[0035] SNP6-R: The nucleotide sequence is as shown in SEQ ID NO: 14.

[0036] Based on this, an embodiment of the present application provides a nucleic acid molecule related to antibacterial sclerotinia and / or anthracnose of mulberry fruits, and the nucleic acid molecule includes the aforementioned molecular marker.

[0037] In some embodiments, the nucleic acid molecule is a fragment of the ALE2 gene.

[0038] In some embodiments, the nucleic acid molecule is a fragment of the ALE2 gene, and the nucleotide sequence length of the nucleic acid molecule is 5353 - 5453 bp. Among them, the length of the upstream sequence located in the sequence shown in SEQ ID NO: 1 is 50 - 100 bp, and the length of the downstream sequence located in the sequence shown in SEQ ID NO: 1 is 50 - 100 bp.

[0039] In some preferred embodiments, the nucleic acid molecule is a fragment of the ALE2 gene, and the nucleotide sequence length of the nucleic acid molecule is 5253 - 5353 bp. Among them, the length of the upstream sequence located in the sequence shown in SEQ ID NO: 1 is 0 - 50 bp, and the length of the downstream sequence located in the sequence shown in SEQ ID NO: 1 is 0 - 50 bp.

[0040] In some more preferred embodiments, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO: 1, with a length of 5253 bp. Among them, N is A or T, that is, there is an A>T single nucleotide mutation at the 4703 bp of the nucleotide sequence shown in SEQ ID NO: 1.

[0041] Based on this, an embodiment of the present application provides a kit for identifying antibacterial sclerotinia and / or anti-anthracnose mulberry fruits, which includes the aforementioned primer set.

[0042] Based on this, an embodiment of the present application provides a method for identifying antibacterial sclerotinia mulberry fruit and / or anti-anthracnose mulberry fruit varieties, including:

[0043] Using the DNA of the mulberry fruit to be tested as a template, performing PCR amplification on it with the aforementioned primer set; directly distinguishing the genotype and the correlation between the genotype and the mulberry fruit variety through fluorescence signals to identify antibacterial sclerotinia mulberry fruit and / or anti-anthracnose mulberry fruit varieties.

[0044] Furthermore, the genotype includes AA genotype, AT genotype, and TT genotype. Among them, both the AT genotype and the TT genotype are antibacterial sclerotinia and anti-anthracnose mulberry fruit varieties at the same time.

[0045] Based on this, an embodiment of the present application provides the application of the aforementioned molecular marker or the aforementioned primer set or the aforementioned kit or the aforementioned method in mulberry fruit breeding.

[0046] In some embodiments, the mulberry fruit variety is antibacterial sclerotinia mulberry fruit.

[0047] In some embodiments, the mulberry variety is a mulberry resistant to anthracnose.

[0048] In some embodiments, the mulberry variety is a mulberry resistant to both sclerotinia and anthracnose.

[0049] The present application will be further described below in conjunction with more specific embodiments. Of course, the following embodiments should not be construed as limiting the present application.

[0050] 1. Evaluation of the resistance of mulberry varieties to sclerotinia

[0051] As shown in Table 1, in the embodiments of the present application, the resistance of 27 mulberry varieties in the germplasm resource nursery of mulberry trees in Hubei Province to sclerotinia was investigated. For each variety, 3 plants were investigated, 1 branch was randomly investigated for each plant, 1 meter length was investigated for each branch, the total number of fruits and the number of diseased fruits on the 1-meter branch were recorded, and the incidence rate = the number of diseased fruit grains / the total number of fruit grains × 100%.

[0052] Table 1

[0053]

[0054]

[0055] Results: In the embodiments of the present application, the resistance of 27 different varieties to sclerotinia was classified. Among them, the incidence rate within 5% was highly resistant, 6%-10% was moderately resistant, 20%-49% was moderately susceptible, and higher than 49% was highly susceptible; finally, 2 highly resistant mulberry varieties, 7 moderately resistant mulberry varieties, 13 moderately susceptible mulberry resources, and 5 highly susceptible mulberry resources were screened out.

[0056] 2. DNA extraction and whole-genome resequencing of mulberry varieties

[0057] In the embodiments of the present application, pest- and disease-free leaves of the above 27 mulberry varieties were collected respectively, DNA was extracted using the E.Z.N.A. Plant DNA Kit kit, and the quality of DNA extraction was detected by 0.8% agarose gel electrophoresis. At the same time, DNA was quantified using an ultraviolet spectrophotometer for whole-genome resequencing. The sequencing library was prepared using the standard library construction process of the TruSeq DNA PCR-free prep kit reagent of Illumina company, and the library insert fragment was about 450 bp; paired-end sequencing of 2×150 bp was performed on the NovaSeq sequencer; the high-quality data obtained after filtering was aligned to the reference genome using the BWA-MEM (version 0.7.12-r1039) program, and the default parameters were used during the alignment process. The GATK software was used to detect SNP molecular markers.

[0058] Results: (1) Among the 27 samples, the G2 sample had the largest amount of high-quality reads, obtaining 96.93% of high-quality data, and the lowest was the G26 sample, obtaining 95.58% of high-quality data. Among the 27 samples, the G1 and G4 samples had the largest amount of high-quality data length, obtaining 94.58% of high-quality data, and the lowest was the G26 sample, obtaining 93.03% of high-quality data. The above results indicate that the data obtained by sequencing has high quality and can meet the requirements of genome sequencing analysis.

[0059] (2) After obtaining high-quality data in the embodiments of the present application, it was aligned to the published reference genome data of Morus alba. After statistics, it was found that the G10 sample had the highest alignment rate, up to 99.82%, and the lowest was the G19 sample, with an alignment rate of 98.99%. The results show that the alignment efficiency of the 27 samples is relatively high, which is beneficial for subsequent analysis.

[0060] (3) The embodiments of the present application statistically analyzed and compared the sequencing depths of 27 samples. The results show that the sequencing depths of the 27 samples are in the range of 21.84× - 26.94×, and the genome coverage rates are between 84.13% - 89.80%. The results show that they have relatively high sequencing depths, and the genome coverage rates also meet the requirements of subsequent analysis.

[0061] (4) The embodiments of the present application divided the 27 samples into two groups: disease-resistant and disease-susceptible according to the results of disease resistance statistics in Table 1. After comparative analysis of the SNP sites in the samples of these two groups, a total of 22,965 group-specific SNPs were identified. As Figure 1 shown, the number of group-specific SNPs unique to the disease-resistant group is 4,220, and the number of group-specific SNPs unique to the disease-susceptible group is 16,023. After analysis, a total of 117 SNP sites associated with proteins were screened.

[0062] 3. SNaPshot technology verification of SNP sites related to fruit mulberry disease resistance

[0063] Collect pest-free leaves of 19 varieties such as "322", "Y2" and Xianfeng long spike mulberry, extract DNA using the E.Z.N.A. Plant DNA Kit kit, and detect the concentration and integrity for SNaPshot genotyping; use the ABI company's SNaPshot Multiplex Kit for SNPs detection.

[0064] PCR amplification, where the PCR reaction system (20 μl) includes 0.8 μl MgCl 2 (50 mmol·L -1 )、2 μl 10×PCR Buffer (Mg2+ free), 0.5 μl dNTP (10 mmol·L -1 ), 0.5 μl of mixed primers, 1 μl of template DNA, 0.5 μl of Platinum Taq (5 U) and 14.2 μl of ddH 2 O; The PCR reaction program was: pre-denaturation at 95°C for 2 min, denaturation at 95°C for 20 s, annealing at 55°C for 30 s, extension at 72°C for 40 s for 35 cycles, and finally extension at 72°C for 5 min;

[0065] Refer to the SNaPshot technology of ABI Company for PCR product purification. Use the PRISM 3730 sequencer of ABI Company for genotyping; compare the genotyping results and sequencing results of 16 mulberry varieties. If the two are consistent, it can be determined that the typing of this locus is correct, otherwise the typing is incorrect.

[0066] Results: According to the population-specific SNP information found, 10 SNP molecular markers were designed (as shown in Table 2). Using the SNaPshot reaction, compare the genotyping results and sequencing results of SNPs in 16 mulberry varieties such as Xianfeng long-spike mulberry. It was found that the genotyping results of 3 loci, SNP6, SNP7, and SNP8, had an accuracy rate of over 93% compared with the sequencing results, indicating that the typing of these 3 SNPs was correct. These 3 loci were associated with 3 genes, namely XM_024172133.1-0 (receptor-like serine / threonine-protein kinase ALE2), XM_024161837.1-0 (shaggy-related protein kinase epsilon), and XM_010108830.1-0 (probably inactive receptor-like protein kinase At2g46850).

[0067] Table 2

[0068]

[0069] 4. Development and verification of KASP markers

[0070] Design KASP primers based on the SNP loci (SNP6, SNP7, SNP8), and then add FAM or HEX fluorescent adapter sequences to the 5' ends of the two forward primers (F1 and F2) respectively. Use Primer 5.0 for primer design, and perform BLASTN homology comparison of the designed primers in the GenBank NR database to analyze their specificity. The primers were synthesized by Beijing Tsingke Biotechnology Co., Ltd.

[0071] Using Direct-PCR Lysis Buffer (Guangzhou Good Biotechnology Co., Ltd.), DNA was rapidly extracted according to the steps of the bead milling method in the instruction manual: Take 2 cm of leaves and place them in a 2 ml centrifuge tube, add 6 3 mm beads, add 400 μl of Good Biotechnology Direct-PCR Lysis Buffer, use a Retsch MM400 grinder at 30 Hz for 60 s, then centrifuge at 3000 rpm for 3 min, pipette 10 μl of the supernatant into an 8-well PCR tube, and add 290 μl of ddH 2 O, mix well and use for detection;

[0072] Twenty-six mulberry resources were used to detect the effectiveness of KASP primers. Three SNP molecular marker sequences were amplified by PCR. Among them, the PCR reaction system was: 100 ng of DNA, 5 μl of Flu-Arms 2xPCR Mix (Guangzhou Good Biotechnology Co., Ltd.), 0.1 μl of F1 (10 μM), 0.1 μl of F2 (10 μM), 0.3 μl of reverse primer (10 μM), and ddH 2 O was supplemented to 10 μl;

[0073] Detection was carried out using a QuantStudio5 real-time fluorescence quantitative instrument. Among them, the PCR reaction program was: 95 °C for 10 min; 95 °C for 15 s, 61 - 55 °C for 60 s, 10 cycles (decreasing 0.6 °C per cycle); 95 °C for 15 s, 55 °C for 60 s, 36 cycles; 30 °C for 30 s to read the plate;

[0074] The selected effective primers were used to verify the mulberry fruit resources and detect the hybrid F1 population to accelerate the breeding process.

[0075] Results: (1) Three SNP loci (SNP6, SNP7, SNP8) screened according to the SNaPshot reaction in the examples of this application were used to design 3 pairs of KASP specific primers. Information such as the primer sequences and the sizes of the amplification products is shown in Table 3.

[0076] Table 3

[0077]

[0078] (2) The results of detecting the DNA of 26 mulberry resources using 3 pairs of KASP specific primers respectively are shown. Figure 2 Shown is the KASP-SNP genotyping detection result of some samples of the molecular marker primer group, among which Figure 2 A is the detection result of 26 mulberry resources, Figure 2Group B was the detection of a partial hybrid F1 population of Pearl White × Kangqing 10; red represents the AA genotype, blue represents the TT genotype, green represents the heterozygous genotype AT, and "×" represents indistinguishable; from the results shown in the figure, SNP7 and SNP8 could not be successfully genotyped, and SNP6 divided the materials into three parts. The red ones were all more susceptible to sclerotinia, the blue ones were all more resistant to sclerotinia, and the green ones were all more resistant to sclerotinia. The marker results were basically consistent with the phenotypic investigation results of the field plants during the flowering period. Therefore, this marker can be used for the identification of sclerotinia resistance in fruit mulberry. The KASP technology developed for gene SNP6 also had good results in the detection of the hybrid F1 population.

[0079] 5. Further application verification of SNP molecular markers

[0080] The obtained hybrid seeds were placed in a petri dish lined with 5 layers of toilet paper and germinated in a 25°C greenhouse for 5 - 7 days. The germinated seeds were dibbled into a planting pot filled with substrate. Hybrid seedlings at the 4 - 5 leaf stage were inoculated with Colletotrichum mori. The obtained single-spore isolated strain of Colletotrichum aenigma was transferred to a PDA plate and cultivated in a 25°C constant temperature incubator for 5 days, then inoculated into YPSS medium (containing 4 g of yeast powder, 15 g of sucrose, 1 g of KH 2 PO 4 , 0.5 g of MgSO 4 ), and cultivated in a 25°C constant temperature shaking incubator at 180 rpm / min for 5 days to prepare a conidial suspension of 1×10 5 spores / ml. After acupuncture, the whole plant of 3-month-old mulberry hybrid seedlings was spray-inoculated, and about 4 ml of spore solution was sprayed on each plant. After inoculation, the mulberry seedlings were covered with plastic bags to keep them moist and cultured under a 12-hour light-dark cycle at 28°C; 6 days after inoculation of the tested mulberry varieties, the incidence rate was counted. The incidence rate of each plant = the number of diseased leaves per plant / the total number of investigated leaves per plant × 100%.

[0081] DPS (version 7.05) software was used to perform variance analysis on the data, and the least significant difference (LSD) method was used to compare the significant differences between different hybrid seedlings inoculated with Colletotrichum mori (P < 0.05). GraphPad Prism 9.0 (GraphPad Software, CA) was used to draw a bar chart. The KASP detection results of each hybrid seedling were converted into data. × indistinguishable was recorded as 0, red was recorded as 1, green was recorded as 2, and blue was recorded as 3. GraphPad Prism9.0 (GraphPad Software, CA) was used to draw a bar chart.

[0082] Figure 3 This is the disease resistance verification result of the SNP molecular marker provided by the embodiment of this application, whereFigure 3 A, Figure 3 B shows the situation of inoculating Colletotrichum gloeosporioides 3 months after the germination and dibbling of hybrid seedlings. By performing association analysis between the screened SNP6 molecular marker and Colletotrichum gloeosporioides resistance, the results show that the hybrid offspring resistant to Sclerotinia sclerotiorum also have a relatively low incidence rate after inoculation with Colletotrichum gloeosporioides (as shown in Figure 3 C).

[0083] Figure 3 D shows the correlation between the incidence rate and the KASP genotyping results. Among them, red represents the AA genotype, which shows relatively high susceptibility to both Sclerotinia sclerotiorum and Colletotrichum gloeosporioides; blue represents the TT genotype, which shows relatively high resistance to both Sclerotinia sclerotiorum and Colletotrichum gloeosporioides; green represents the heterozygous genotype AT, which also shows relatively high resistance to Sclerotinia sclerotiorum and Colletotrichum gloeosporioides, indicating that the SNP6 molecular marker can also be used as a marker for Colletotrichum gloeosporioides resistance.

[0084] In summary, the Sclerotinia sclerotiorum - resistant SNP molecular marker of mulberry fruits developed based on the KASP technology provided in the embodiments of the present application can not only be used for the identification of Sclerotinia sclerotiorum resistance of mulberry fruits, but also for the identification of Colletotrichum gloeosporioides resistance of mulberry fruits. Based on the KASP technology, SNP genotyping can be accurately performed, greatly improving the selection of materials for Sclerotinia sclerotiorum and Colletotrichum gloeosporioides resistance of mulberry fruits, and greatly shortening the breeding cycle of mulberry varieties. Its detection is convenient, fast, has a high throughput, low cost and is not affected by the environment.

[0085] The above is only a preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed in the present application should be covered by the protection scope of the present application.

Claims

1. SNP molecular markers related to fruit mulberry resistant to Sclerotinia sclerotiorum, characterized in that, the SNP molecular marker is the nucleotide sequence shown in SEQ ID NO:

1. In the nucleotide sequence shown in SEQ ID NO:1, N is A or T, and the N is located at the 18,208,100th nucleotide site on chromosome No. 4 of the white mulberry genome.

2. A primer set for amplifying the SNP molecular marker according to claim 1, characterized in that, the primer set is: SNP6-F1: the nucleotide sequence is as shown in SEQ ID NO:12; SNP6-F2: the nucleotide sequence is as shown in SEQ ID NO:13; SNP6-R: the nucleotide sequence is as shown in SEQ ID NO:

14.

3. A kit for identifying fruit mulberry resistant to Sclerotinia sclerotiorum and / or Colletotrichum gloeosporioides, characterized in that, the kit includes the primer set according to claim 2.

4. A method for identifying fruit mulberry varieties resistant to Sclerotinia sclerotiorum and / or Colletotrichum gloeosporioides, characterized in that, the method includes: using the DNA of the fruit mulberry to be tested as a template, and performing PCR amplification on it with the primer set according to claim 2; directly distinguishing the genotype and the correlation between the genotype and the fruit mulberry variety through fluorescence signals to identify fruit mulberry varieties resistant to Sclerotinia sclerotiorum and / or Colletotrichum gloeosporioides.

5. The method according to claim 4, characterized in that, the genotype includes AA genotype, AT genotype and TT genotype. Among them, both the AT genotype and the TT genotype are fruit mulberry varieties resistant to Sclerotinia sclerotiorum and Colletotrichum gloeosporioides at the same time.

6. Application of the molecular marker according to claim 1, or the primer set according to claim 2, or the kit according to claim 3, or the method according to any one of claims 4 to 5 in fruit mulberry breeding.

7. The application according to claim 6, characterized in that, the fruit mulberry variety is fruit mulberry resistant to Sclerotinia sclerotiorum.

8. The application according to claim 6, characterized in that, the fruit mulberry variety is fruit mulberry resistant to Colletotrichum gloeosporioides.

9. The application according to claim 6, characterized in that, the fruit mulberry variety is fruit mulberry resistant to both Sclerotinia sclerotiorum and Colletotrichum gloeosporioides.

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