SNP molecular marker and application thereof in regulating cold resistance of malus plant

Abstract

This invention relates to the field of plant breeding technology, and more particularly to an SNP molecular marker and its application in regulating cold resistance in *Malus* species. Based on the genome version *Msieversii_haploid_v2*, the SNP molecular marker includes SNP1 and / or SNP2. The applications include: (1) detecting cold resistance in *Malus* species; (2) breeding highly cold-resistant *Malus* varieties; (3) marker-assisted breeding of *Malus* species; (4) genome-wide association analysis of *Malus* species; (5) improvement of *Malus* varieties related to cold resistance; and (6) improvement of *Malus* germplasm resources. This invention has screened and obtained SNP molecular markers related to cold resistance in *Malus* species, which can be used for detecting cold resistance in *Malus* species and for breeding highly cold-resistant *Malus* varieties, thus having significant application value in the field of *Malus* breeding.

Description

Technical Field

[0001] This invention relates to the field of plant breeding technology, and in particular to an SNP molecular marker and its application in regulating the cold resistance of Malus species. Background Technology

[0002] Xinjiang wild apple ( Malus sieversii Rich in stress-resistant genetic resources, cold resistance is its core superior trait, making it an important gene donor for cold-resistant apple breeding. This is of great significance for cultivating new cold-resistant apple varieties, expanding the apple planting area, and reducing industrial losses caused by low-temperature disasters. Low-temperature freezing damage, as a common abiotic stress, easily leads to frost damage, wilting, and even death of wild apple plants, resulting in yield losses. Therefore, cultivating cold-resistant varieties and mitigating the damage from freezing has become a key research direction for improving the safety and economic value of the apple industry.

[0003] SNP (single nucleotide polymorphism) molecular markers have advantages such as wide distribution, high stability and rapid detection. They are the core technical means to elucidate the molecular mechanism of plant stress resistance and realize the rapid screening of stress-resistant germplasm. Gene chips developed based on SNP molecular markers can realize simultaneous and efficient detection of multiple sites, which greatly improves the efficiency of breeding screening. They have been widely used in research on plant stress resistance traits and molecular marker-assisted breeding.

[0004] Several SNP loci related to cold resistance in *Malus* species have been reported. Some of these SNP loci were obtained using SLAF-seq simplified genome sequencing technology and have been confirmed to be related to physiological processes in which plants respond to abiotic stresses such as cold resistance. Additionally, SNP markers and corresponding primers related to cold resistance in apple rootstocks have been reported, which can be used for preliminary identification of cold resistance in apple rootstocks. However, existing research still has significant limitations. The molecular regulatory basis of cold resistance traits in wild apple remains unclear, and there is a lack of efficient and precise SNP molecular marker combinations and corresponding detection tools specifically for screening cold-resistant wild apple germplasm from Xinjiang.

[0005] Furthermore, cold resistance in Xinjiang wild apples, as a typical quantitative trait controlled by multiple genes, exhibits a characteristic of small-scale cumulative effects of multiple genes in its genetic regulation. Currently, Xinjiang wild apples face challenges such as insufficient analysis of the genetic regulatory network for cold resistance and an unexplained multi-gene interaction mechanism. These limitations make it difficult to achieve effective improvement of cold resistance traits and precise screening of cold-resistant germplasm using traditional molecular marker methods based on single or a few SNP loci. Therefore, developing combinatorial markers and high-throughput detection tools (such as gene chips) that can cover multiple key loci and reflect the cumulative effects of multiple genes, thereby enabling efficient and accurate evaluation of cold-resistant germplasm in Xinjiang wild apples, has become a crucial technical problem urgently needing to be solved in the field of wild apple genetics and breeding. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides an SNP molecular marker and its application in regulating the cold resistance of Malus species.

[0007] In a first aspect, the present invention provides an SNP molecular marker, including: SNP1 and / or SNP2; Based on the genome version Msieversii_haploid_v2, the genomic location of SNP1 is Chr10: 23043489 bp, and the polymorphism is A / G; The SNP2 is located at Chr11:14023884 bp in the genome, and its polymorphism is C / A.

[0008] The position information used by each SNP molecular marker in this invention is in the conventional and defined form in the art. For example, Chr10: 23043489 bp, where Chr10 represents the position at 23043489 bp on chromosome 10.

[0009] The present invention further provides a molecular marker, including: an SNP1 marker and / or an SNP2 marker; The SNP1 marker includes a nucleotide sequence as shown in SEQ ID NO.1, with a polymorphism at position 25, which is A / G. The SNP2 marker includes a nucleotide sequence as shown in SEQ ID NO.2, with a polymorphism at position 25, which is C / A. The nucleotide sequence shown in SEQ ID NO.1 is: 5'-AATATAGAGGCATTATCCAAGTTTAGTGATTTATGACTGTAAACCCTAAA-3'.

[0010] The nucleotide sequence shown in SEQ ID NO.2 is: 5'-CGAACAACCAATTCGGGTTTATGCCAGGACGGTCAACCATGGAGGCAATC-3'.

[0011] Furthermore, the present invention provides a combination of SNP molecular markers, including both SNP1 and SNP2, to further improve the accuracy of breeding related to cold resistance in Malus species.

[0012] Secondly, the present invention provides a primer pair for amplifying the aforementioned SNP molecular marker.

[0013] The primer pair design method described in this invention can be a conventional method of this invention. Technicians can design primer pairs (including primer pairs or KASP primer combinations) of different lengths based on existing primer design rules and primer design software (such as Primer) to amplify the aforementioned molecular markers.

[0014] Thirdly, the present invention provides a probe for detecting the aforementioned SNP combinations.

[0015] Fourthly, the present invention provides a gene chip including the aforementioned probes.

[0016] Preferably, the gene chip is a solid-phase in-situ synthesis chip, a solid-phase bead array chip, a liquid-phase capture chip, or a targeted sequencing genotyping chip.

[0017] Fifthly, the present invention provides a kit comprising the aforementioned SNP molecular marker, or the aforementioned primer pair, or the aforementioned probe, or the aforementioned gene chip.

[0018] In a sixth aspect, the present invention provides the application of the aforementioned SNP-labeled detection reagent, or the aforementioned primer pair, or the aforementioned probe, or the aforementioned gene chip in any of the following: (1) To detect the cold resistance of apple plants, or to prepare reagents for detecting the cold resistance of apple plants; (2) To cultivate highly cold-resistant apple varieties, or to prepare reagents for cultivating highly cold-resistant apple varieties; (3) Molecular marker-assisted breeding of apple species; (4) Genome-wide association analysis of apple species; (5) Improvement of apple varieties related to cold resistance; (6) Germplasm resource improvement of apple species.

[0019] In a seventh aspect, the present invention provides a method for detecting the cold resistance of apple plants, comprising: detecting the polymorphism of the aforementioned SNP molecular markers in the apple plant to be tested, and determining the cold resistance of the apple plant to be tested based on the genotype detection results.

[0020] Furthermore, the determination of the cold resistance of the tested *Malus* plant based on the genotype detection results includes: For SNP1, plants with a genotype of GG have higher cold resistance than plants with a genotype of AA. For SNP2, plants with a genotype of AA have higher cold resistance than plants with a genotype of CC.

[0021] This invention characterizes the cold resistance of wild apples using the Pt / St ratio. For SNP1, plants with a genotype of GG have a much higher Pt / St ratio than plants with a genotype of AA. Similarly, for SNP2, plants with a genotype of AA have a much higher Pt / St ratio than plants with a genotype of CC.

[0022] Eighthly, the present invention provides a method for breeding varieties of *Malus* plants, comprising: during the breeding process of *Malus* plants, selecting offspring that conform to any of the following genotypes from the aforementioned SNP molecular markers: (1) The genotype of SNP1 is GG; (2) The genotype of SNP2 is AA.

[0023] Furthermore, the apple species include one or more of the following: Xinjiang wild apple, European wild apple, Malus baccata, or Malus baccata, preferably Xinjiang wild apple.

[0024] The present invention has the following beneficial effects: This invention screened and identified SNP molecular markers related to cold resistance in *Malus* species, revealing the genetic variation characteristics of cold resistance traits at the haplotype level. This enables precise identification of cold-resistant genotypes in *Malus* germplasm resources, providing a novel tool for marker-assisted selection breeding. Furthermore, the cold resistance-related haplotype markers obtained in this invention can be used for further development of gene chips targeting cold resistance-related loci in *Malus* species.

[0025] The SNP molecular markers provided by this invention can be applied to cultivate highly cold-resistant apple varieties, which has important application value in the field of apple breeding. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0027] Figure 1 This is a statistical result and distribution diagram of the ratio of palisade tissue thickness to spongy tissue thickness in wild apple plants provided in this embodiment of the invention.

[0028] Figure 2 This is a Manhattan plot of GWAS analysis of the cold resistance trait of Xinjiang wild apple provided in an embodiment of the present invention.

[0029] Figure 3 This is the Chr10: 23043489 bp site provided in this embodiment of the invention (corresponding to the encoding TLP1 protein). Msi_ 10g012570 Expression profiles of genes.

[0030] Figure 4 This is the Chr11: 14023884 bp site provided in this embodiment of the invention (corresponding to the encoding of the MLP43 protein). Msi_ 11g012390 Expression profiles of genes.

[0031] Figure 5 This is the Chr10: 23043489 bp site provided in this embodiment of the invention (corresponding to the encoding TLP1 protein). Msi_ 10g012570 Gene structure and haplotype analysis diagram of the gene.

[0032] Figure 6 This is the Chr10: 23043489 bp site provided in this embodiment of the invention (corresponding to the encoding GRP2 protein). Msi_ 10g012540 Gene structure and haplotype analysis diagram of the gene.

[0033] Figure 7 This is the Chr11: 14023884 bp site provided in this embodiment of the invention (corresponding to the encoding of the MLP43 protein). Msi_ 11g012390 Gene structure and haplotype analysis diagram of the gene.

[0034] Figure 8 This is the association analysis result of the cold resistance index (Pt / St ratio) of different haplotypes of Xinjiang wild apple at the Chr10:23043489 bp site provided in the embodiments of the present invention.

[0035] Figure 9 This is the association analysis result of the cold resistance index (Pt / St ratio) of different haplotypes of Xinjiang wild apple at the Chr11:14023884 bp site provided in the embodiments of the present invention. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0037] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0038] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0039] Example 1: Genome-wide association analysis of cold resistance traits in Xinjiang wild apples and verification at Chr10: 23043489 and Chr11: 14023884 loci. (1) Phenotypic acquisition.

[0040] In the natural distribution areas of *Prunus cerasifera* in Tuoli County and Gongliu County, Xinjiang, 100 *Prunus cerasifera* trees were randomly selected and their leaves were collected. These 100 trees were arranged in a completely randomized block design, with a minimum spacing of 50 meters between each tree to avoid interference from genetic associations between trees. After collection, all leaf samples were paraffin-embedded, and the thickness of the palisade tissue (Pt) and spongy tissue (St) was observed and measured using an optical microscope. The Pt / St ratio (the ratio of palisade tissue thickness to spongy tissue thickness) was calculated based on the measured thickness data, and a normality test was performed. The results are as follows: Figure 1 As shown.

[0041] (2) Genotype identification.

[0042] One hundred leaf samples from *Prunus cerasifera* (Xinjiang wild apple) were collected, and genomic DNA was extracted using the CTAB method. Whole-genome sequencing was then performed using a high-throughput sequencing platform. Raw sequencing data were quality filtered using FastP v0.23.2 software. The filtered valid data were then aligned to the *Prunus cerasifera* reference genome *Msieversii_haploid_v2* using BWA software, removing secondary alignments and supplementary alignment sequences. PCR repetitive sequences were removed using Picard Tools software. Variants were detected in individual samples using the HaplotypeCaller module of GATK v4.6.1.0 software, generating GVCF files. These GVCF files were integrated into GenomicsDB using GenomicsDBImport, and then combined with GenotypeGVCFs for multi-sample genotyping. Linkage disequilibrium screening was performed using Plink 1.9 software, ultimately obtaining 689,564 SNP markers covering the entire genome.

[0043] (3) Association analysis.

[0044] Multidimensional scaling (MDS) and ADMIXTURE software were used to predict and analyze population structure and potential phylogenetic relationships. Genome-wide association analysis (GWAS) was conducted using a compressed mixed linear model (CMLM) in GAPIT software. Based on phenotypic and genotypic data, the association between cold resistance traits and molecular markers in Xinjiang wild apples was analyzed. Manhattan plots were generated using the R package CMplotv3.6.0 (https: / / github.com / YinLiLin / R-CMplot) to illustrate the association between the Pt / St ratio and SNP loci in the GWAS analysis results of the cold resistance trait (Pt / St). Figure 2 The horizontal axis represents the physical location of the SNP locus on chromosomes 1–17, and the vertical axis represents the statistical significance intensity, expressed as -log10 (p-value). A larger p-value indicates a smaller p-value and a higher probability of association between the SNP locus and the cold resistance trait. A QQ plot was also plotted. The results show that the curve's starting point is basically on the diagonal. When the p-value is greater than 3, the locus begins to deviate from the straight line and curves upward, indicating that false positives and false negatives are well controlled. This demonstrates that the CMLM model used is suitable for the genome-wide association analysis of the cold resistance trait in this study.

[0045] (4) Prediction of functional genes.

[0046] Based on the results of the mixed linear model analysis, the SNP at position 23043489 on chromosome 10 was significantly associated with cold resistance in *Prunus cerasifera* (p-value = 2.125156e-07), and the SNP at position 14023884 on chromosome 11 was also significantly associated with cold resistance in *Prunus cerasifera* (p-value = 3.969755e-07). Using the *Prunus cerasifera* reference genome sequence as a basis, three candidate genes potentially involved in the regulation of cold resistance in *Prunus cerasifera* were screened within a 20kb range upstream and downstream of the two significantly associated SNP sites. Msi_10g012570 , Msi_10g012540 and Msi_11g012390 After subjecting Xinjiang wild apple plants to 0h, 8h, and 72h of low-temperature stress, RNA-Seq technology was used for sequencing to obtain the expression profiles of two candidate genes. Specific results are shown in [link to results]. Figure 3 , Figure 4 .in, Msi_10g012570 The gene function annotation identifies it as encoding the TLP1 protein (a sweet protein). This protein acts as a positive regulator of low-temperature stress response. Under low-temperature conditions, it can significantly enhance the antifreeze ability of Xinjiang wild apple cells by stabilizing cell membrane structure, scavenging reactive oxygen species, and inhibiting ice crystal growth. It belongs to the disease-related protein family with stress resistance functions. Msi_10g012540The gene function annotation identifies it as encoding the GRP2 protein (a glycine-rich RNA-binding protein). This protein is specifically expressed in response to low temperatures and can regulate the RNA metabolism of low-temperature response genes through its cold shock domain, thereby maintaining the homeostasis of plant gene expression under low-temperature conditions and effectively enhancing the frost tolerance of Xinjiang wild apples. Msi_ 11g012390 The gene function annotation identifies it as encoding the MLP43 protein (an actin-binding protein). This protein can promote the accumulation of antifreeze substances by regulating the dynamic stability of the cytoskeleton and the cold resistance signal transduction pathway, thus protecting the integrity of cell structure under low temperature stress. It is a key protein in the core regulatory network of cold resistance traits in Xinjiang wild apples.

[0047] Example 2 developed haplotype markers for the cold resistance trait associated loci in Example 1. (1) Based on the major associated loci Chr10: 23043489 and Chr11: 14023884 of the cold resistance trait in wild apple obtained in Example 1, SNP genotype information of the upstream and downstream 20 kb regions of these two candidate associated loci was obtained. Among them, 36 high-quality SNP variant loci were screened out of Chr10: 23043489, and 218 high-quality SNP variant loci were screened out of Chr11: 14023884. Figures 5-7 ).

[0048] (2) The high-quality SNP genotype information of the above-mentioned wild apple natural population was used to perform linkage disequilibrium (LD) analysis using the software TASSEL5, and the PairWise R between each SNP locus was calculated. 2 The value (between 0 and 1, representing the degree of linkage between two SNPs, with 1 indicating complete linkage) was used to identify 9 closely linked SNPs for Chr10: 23043489. Details are shown in Table 1. Similarly, for Chr11: 14023884, 18 closely linked SNPs were identified. Details are shown in Table 2.

[0049] Table 1. SNPs closely linked to Chr10: 23043489

[0050] Table 2. SNPs closely linked to Chr11: 14023884

[0051] Example 3: Population validation of the correlation between molecular markers and cold resistance traits (1) Genotype testing.

[0052] In addition, a total of 67 wild apple plants from Xinjiang were randomly selected. Genotyping was performed on two SNP molecular markers, Chr10:23043489 and Chr11:14023884, to determine the allele genotype of each plant at the above loci.

[0053] (2) Determination of cold resistance phenotype and correlation statistical analysis.

[0054] The ratio of palisade tissue thickness to spongy tissue thickness (Pt / St) in leaves was determined using the same method as in Example 1 and used as an indicator of cold resistance. Cold resistance phenotypic data were statistically analyzed for homozygous genotypes of each molecular marker, and analysis of variance and multiple comparisons were performed. The results are as follows: Chr10: 23043489 loci: e.g. Figure 8 As shown, haplotype Hap2_G / Hap2_G has a higher Pt / St value than Hap1_A / Hap1_A, which means it has higher cold resistance. The difference is highly significant (P < 0.001), indicating that GG is a cold-resistant dominant genotype that can significantly improve the cold resistance of Xinjiang wild apple.

[0055] Chr11:14023884 site: as shown Figure 9 As shown, haplotypes Hap1_A / Hap1_A have higher Pt / St values ​​than Hap2_C / Hap2_C, which means they have higher cold resistance. The difference is highly significant (P < 0.01), indicating that AA is a cold-resistant dominant genotype that can significantly improve the cold resistance of wild apples in Xinjiang.

[0056] The above results further verify that the molecular markers Chr10:23043489 and Chr11:14023884 obtained in this invention have a stable and significant correlation with the cold resistance trait of Xinjiang wild apple. The differences in cold resistance phenotypes among different genotypes are statistically significant and can be effectively used for the identification of cold-resistant germplasm of Xinjiang wild apple and molecular marker-assisted breeding.

[0057] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An SNP molecular marker, characterized in that, include: SNP1 and / or SNP2; Based on the genome version Msieversii_haploid_v2, the genomic location of SNP1 is Chr10:23043489bp, and the polymorphism is A / G; The SNP2 is located at Chr11:14023884 bp in the genome, and its polymorphism is C / A.

2. A molecular marker, characterized in that, include: SNP1 and / or SNP2 tags; The SNP1 marker includes the nucleotide sequence shown in SEQ ID NO.1, with a polymorphism at position 25, which is A / G. The SNP2 marker includes a nucleotide sequence as shown in SEQ ID NO.2, with a polymorphism at position 25, which is C / A.

3. A primer pair, characterized in that, The primer pair is used to amplify the SNP molecular markers described in claim 1 or 2.

4. A probe, characterized in that, The probe is used to detect the SNP combination as described in claim 1 or 2.

5. A gene chip, characterized in that, Includes the probe described in claim 4.

6. A reagent kit, characterized in that, This includes the SNP molecular marker as described in claim 1 or 2, or the primer pair as described in claim 3, or the probe as described in claim 4, or the gene chip as described in claim 5.

7. The use of the SNP-labeled detection reagent of claim 1 or 2, or the primer pair of claim 3, or the probe of claim 4, or the gene chip of claim 5, in any of the following: (1) To detect the cold resistance of apple plants, or to prepare reagents for detecting the cold resistance of apple plants; (2) To cultivate highly cold-resistant apple varieties, or to prepare reagents for cultivating highly cold-resistant apple varieties; (3) Molecular marker-assisted breeding of apple species; (4) Genome-wide association analysis of apple species; (5) Improvement of apple varieties related to cold resistance; (6) Germplasm resource improvement of apple species.

8. A method for detecting the cold resistance of apple plants, characterized in that, include: The polymorphism of the SNP molecular marker as described in claim 1 or 2 is detected in the test apple species, and the cold resistance of the test apple species is determined based on the genotype detection results.

9. The method according to claim 8, characterized in that, The determination of the cold resistance of the tested Malus species based on genotype testing results includes: For SNP1, plants with a genotype of GG have higher cold resistance than plants with a genotype of AA. For SNP2, plants with a genotype of AA have higher cold resistance than plants with a genotype of CC.

10. A method for breeding varieties of apple plants, characterized in that, include: In the breeding process of Malus species, offspring that conform to any of the following genotypes among the SNP molecular markers described in claim 1 or 2 are selected: (1) The genotype of SNP1 is GG; (2) The genotype of SNP2 is AA.

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