SSR (Simple Sequence Repeat) molecular marker for screening nuclear cracking resistance character of peach and application of SSR molecular marker

Abstract

The invention discloses an SSR molecular marker for screening nuclear cracking resistance characters of peaches and application of the SSR molecular marker, and relates to the technical field of genetic breeding of fruit trees, the SSR molecular marker is named as PpCPK12-SSR, and the nucleotide sequence of the SSR molecular marker is shown as SEQ ID NO.1. The invention also provides a specific primer pair for amplifying the marker, the nucleotide sequence of an upstream primer of the specific primer pair is shown as SEQ ID NO.2, and the nucleotide sequence of a downstream primer of the specific primer pair is shown as SEQ ID NO.2. The nucleotide sequence of the downstream primer is as shown in SEQ ID NO.3. Through PCR (polymerase chain reaction) amplification and electrophoresis detection, according to the size of an amplified fragment, the anti-nuclear-cleavage genotype of the peach can be quickly and accurately identified in the seedling stage of the peach: an individual carrying a 115bp fragment is an anti-nuclear-cleavage type; the defects that traditional field phenotype identification is long in period, low in efficiency and affected by the environment are overcome, an efficient molecular marker auxiliary selection tool is provided for peach nuclear cracking resistance breeding, and the breeding process can be remarkably accelerated.

Description

Technical Field

[0001] This invention relates to the field of fruit tree genetics and breeding technology, specifically to an SSR molecular marker for screening peach pit cracking resistance and its application. Background Technology

[0002] Peaches are one of my country's important economic fruit trees, and their fruit quality and yield directly affect economic benefits. Pit cracking is a common physiological disorder in peach production, mainly manifested as the pit (inner pericarp) cracking during the later stages of fruit development. Although the appearance of cracked fruit may not be affected, the pit is prone to mold growth, leading to poor fruit development, easy fruit drop, poor storage and transportation quality, and a significant reduction in commercial value.

[0003] Currently, screening for peach pit crack resistance mainly relies on field phenotypic identification. Breeders need to dissect each fruit during ripening to observe whether the pit cracks, but this method has the following inherent drawbacks:

[0004] 1. Long cycle and low efficiency: Identification can only be carried out when the fruit is ripe, which consumes a lot of manpower, material resources and time.

[0005] 2. Highly affected by the environment: The splitting trait is affected by factors such as climate (e.g., water stress) and cultivation management, resulting in unstable phenotypes, which may lead to misselection or omission.

[0006] 3. Inability to select early: It is impossible to screen seedlings or before flowering, resulting in a large breeding population and increased field management and breeding costs.

[0007] Marker-assisted selection (SSR) technology provides an effective approach to overcome the aforementioned challenges. SSR markers are widely used in plant genetics and breeding due to their advantages such as wide distribution in the genome, high polymorphism, co-dominant inheritance, and ease of operation. However, there are currently no publicly reported SSR molecular markers that are stably linked to the peach's resistance to pit splitting and are suitable for large-scale breeding screening.

[0008] Therefore, developing an SSR molecular marker that can accurately, rapidly, and early identify peach seed-cracking resistance is of vital importance for accelerating the peach seed-cracking resistance breeding process. Summary of the Invention

[0009] Technical problem solved: In response to the technical shortcomings of existing technologies, such as long cycle, low efficiency, great susceptibility to environmental influence, inability to select early and poor accuracy caused by reliance on phenotypic identification, a method for SSR molecular markers for screening peach kernel crack resistance is disclosed.

[0010] Purpose of the invention: The purpose of this invention is to provide an SSR molecular marker closely linked to the peach seed-cracking resistance trait and its application, and to provide the application of the above-mentioned SSR molecular marker in marker-assisted selection breeding of peach seed-cracking resistance trait.

[0011] Technical solution:

[0012] An SSR molecular marker for screening peaches for resistance to pit splitting, the SSR molecular marker is named PpCPK12-SSR, and its nucleotide sequence is shown in SEQ ID NO.1: GGCGGCGGCGGCGGCGGCGGCGGCGGCGCTCCGAGCTCGGATTTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTCACACACACACACACACACACACACACACACACACACACACACACACACACACATCTAGCTGAATTCGATC.

[0013] The present invention also provides a primer pair for amplifying the SSR molecular marker for screening peach kernel-resistant traits, the primer pair comprising an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown in SEQ ID NO.2: 5'-ATGGCTCCGAGCTCGGATTT-3', and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.3: 5'-GATCGATTCAGCTAGATGTG-3'.

[0014] This application also discloses a kit for screening peaches for resistance to pit cracking, the kit containing the primer pairs described above.

[0015] This invention further provides the application of the SSR molecular marker in the identification of crack-resistant traits in peach germplasm resources.

[0016] The above-mentioned SSR molecular markers for screening peach seed crack resistance, the above-mentioned primer pairs, or the above-mentioned kits are used in peach molecular marker-assisted selection breeding.

[0017] A molecular detection method for peach pit cracking resistance includes the following steps:

[0018] Step a: Extract genomic DNA from the peach sample to be tested;

[0019] Step b: Using the DNA extracted in step a as a template, perform PCR amplification using the upstream primer shown in SEQ ID NO.2 and the downstream primer shown in SEQ ID NO.3;

[0020] Step c: Detect the fragment size of the PCR amplification product;

[0021] Step d: Determine the genotype of the peach sample to be tested for the trait of nucleus cracking based on the fragment size: when the amplified fragment is 115bp, it is determined to be the homozygous genotype of nucleus cracking resistance AA; when fragments of 115bp and 223bp are amplified at the same time, it is determined to be the heterozygous genotype of nucleus cracking resistance AB; when the amplified fragment is 223bp, it is determined to be the homozygous genotype of nucleus cracking susceptibility BB.

[0022] Furthermore, in step c, the method for detecting the fragment size of the PCR amplification product is electrophoresis.

[0023] This application presents an SSR molecular marker for screening peach pit cracking resistance and its application, which has the following advantages compared with existing technologies:

[0024] 1. High accuracy: The SSR molecular marker PpCPK12-SSR provided by this invention co-segregates with the peach kernel splitting resistance trait, which can accurately reflect the individual's genotype and is not affected by environmental factors;

[0025] 2. Early selection: Testing can be carried out during the seedling stage of peach trees (after the leaves unfold), without waiting for flowering and fruiting, which significantly shortens the breeding cycle (by 3-4 years).

[0026] 3. Simple operation and low cost: SSR marker technology is mature, the detection process is simple, and it is easy to promote and apply in routine molecular biology laboratories. It is suitable for rapid screening of large-scale breeding populations.

[0027] 4. High efficiency: By using the markers of this invention for assisted selection, individuals that do not carry resistance alleles can be eliminated early, and resources can be concentrated to cultivate superior single plants, which greatly improves breeding efficiency. Attached Figure Description

[0028] Figure 1 This is a gel electrophoresis image of the PCR amplification products of this application. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0030] Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and the experimental methods without specific conditions are all conventional methods in the art.

[0031] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0032] Example 1, the acquisition of SSR molecular markers and primer design, are as follows:

[0033] Bioinformatics analysis of gene regions related to cell wall development and calcium signal transduction in the peach reference genome revealed a highly polymorphic SSR site within a calcium-dependent protein kinase gene located on chromosome 5. Based on its flanking conserved sequence, specific primers were designed using Primer Premier 6.0 software. The upstream primer sequence is shown in SEQ ID NO.2: 5'-ATGGCTCCGAGCTCGGATTT-3', and the downstream primer sequence is shown in SEQ ID NO.3: 5'-GATCGATTCAGCTAGATGTG-3'. The expected amplified fragment length is between 115-223 bp.

[0034] The SSR molecular marker is named PpCPK12-SSR, and its nucleotide sequence is shown in SEQ ID NO.1: GGCGGCGGCGGCGGCGGCGGCGGCGGCGCTCCGAGCTCGGATTTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTCACACACACACACACACACACACACACACACACACACACACACACACACACACACATCTAGCTGAATTCGATC.

[0035] Example 2: Genotypic identification and phenotypic verification of peach varieties:

[0036] 1. Experimental materials:

[0037] Thirty peach varieties with known split-kernel phenotypes were selected as experimental materials, including 10 varieties known to be resistant to split-kernel (such as 'Zhongtao Kanglie No. 1'), 10 varieties known to be split-kernel (such as 'Dajiubo'), and 10 varieties with unknown phenotypes.

[0038] 2. Experimental methods:

[0039] DNA extraction: Genomic DNA was extracted from young leaves of various varieties using the CTAB method.

[0040] PCR amplification: Reaction system (20μL): 10μL 2×Taq PCR Master Mix, 0.5μL each of forward and reverse primers (10μM), 1μL template DNA (50ng / μL), and 8μL ddH2O.

[0041] PCR program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; 72℃ final extension for 5 min.

[0042] Electrophoresis detection: Take 5 μL of PCR product, separate it using 8% non-denaturing polyacrylamide gel electrophoresis, observe and record the band size after silver staining.

[0043] 3. Results and Analysis:

[0044] Electrophoresis results showed that all known seed-resistant varieties exhibited only one band of approximately 115 bp (AA type); all known seed-splitting varieties exhibited only one band of approximately 223 bp (BB type). Of the 10 varieties with unknown phenotypes, 3 were AA type, 5 were AB type, and 2 were BB type. Phenotypic validation was performed at subsequent fruit ripening stages. AA and AB type individuals showed seed-resistant characteristics, while BB type individuals showed seed-splitting characteristics, validating the marker's accuracy as 100%.

[0045] Experimental verification data: To verify the reliability of the SSR marker of the present invention, this application conducted repeatability experiments and expanded population verification, as shown in Table 1.

[0046] Table 1: Correspondence between PpCPK12-SSR marker genotypes and split-kernel phenotypes in some peach varieties

[0047] Variety Name Marker genotype Predictive phenotype Actual nucleus splitting rate (%) Phenotypic determination Zhongtao Anti-Crack No. 1 AA anti- 2.5% Fragmentation-resistant nuclei Zhongtao Anti-cracking No. 2 AA anti- 3.1% Fragmentation-resistant nuclei Okubo BB feel 85.6% Sensory nucleus Bai Feng BB feel 78.9% Sensory nucleus Spring Snow AB anti- 5.3% Fragmentation-resistant nuclei Autumn Snow AB anti- 4.7% Fragmentation-resistant nuclei Unknown variety 1 AA anti- 4.0% Fragmentation-resistant nuclei Unknown variety 2 BB feel 82.5% Sensory nucleus

[0048] Conclusion: The SSR molecular marker PpCPK12-SSR and its detection primers of the present invention can be stably and accurately used for molecular identification of peach kernel cracking resistance, and are completely consistent with the field phenotypic identification results. They can play an important role in peach molecular marker-assisted breeding.

[0049] The embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. The scope of protection of the present invention is determined by the scope claimed in the claims. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. An SSR molecular marker for screening peach pit crack resistance, characterized in that, The SSR molecular marker is named PpCPK12-SSR, and its nucleotide sequence is shown in SEQ ID NO.1: GGCGGCGGCGGCGGCGGCGGCGGCGGCGCTCCGAGCTCGGATTTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTCACACACACACACACACACACACACACACACACACACACACACACACACACACACATCTAGCTGAATTCGATC.

2. The primer pair used to amplify the SSR molecular marker for screening peach kernel crack resistance as described in claim 1, characterized in that, The primer pair includes an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown in SEQ ID NO.2: 5'-ATGGCTCCGAGCTCGGATTT-3', and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.3: 5'-GATCGATTCAGCTAGATGTG-3'.

3. A kit for screening peaches for resistance to pit cracking, characterized in that, The kit contains the primer pair as described in claim 2.

4. The application of the SSR molecular marker as described in claim 1 in the identification of crack-resistant traits in peach germplasm resources.

5. The application of an SSR molecular marker as described in claim 1, a primer pair as described in claim 2, or a kit as described in claim 3 in marker-assisted selection breeding of peaches.

6. A molecular detection method for peach pit cracking resistance, comprising the following steps: Step a: Extract genomic DNA from the peach sample to be tested; Step b: Using the DNA extracted in step a as a template, perform PCR amplification using the primer pair described in claim 2; Step c: Detect the fragment size of the PCR amplification product; Step d: Determine the genotype of the peach sample to be tested for the trait of nucleus cracking based on the fragment size: when the amplified fragment is 115bp, it is determined to be the homozygous genotype of nucleus cracking resistance AA; when fragments of 115bp and 223bp are amplified at the same time, it is determined to be the heterozygous genotype of nucleus cracking resistance AB. When the amplified fragment is 223bp, it is determined to be the homozygous genotype BB with a split nucleus.

7. The molecular detection method according to claim 6, characterized in that: In step c, the method for detecting the fragment size of the PCR amplification product is electrophoresis.

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