Chinese rose ethylene-sensitive exfoliated whole genome SNP molecular marker combination and application thereof

Abstract

The invention discloses a Chinese rose ethylene-sensitive exfoliated whole genome SNP (Single Nucleotide Polymorphism) molecular marker combination and application thereof, and solves the problems that the traditional Chinese rose breeding depends on field phenotypic observation, has the defects of low efficiency, long period and the like, and is difficult to meet the modern breeding requirements. According to the invention, through whole genome sequencing and association analysis, 528 SNP markers significantly associated with the ethylene-sensitive shedding of the Chinese rose are screened, and the markers are widely distributed in the genome of the Chinese rose and have high significance. A whole genome selection model constructed based on the SNP markers shows excellent prediction accuracy. By utilizing the SNP molecular marker combination, the probe or the chip, the early prediction of the ethylene-sensitive exfoliation character of the Chinese rose can be realized, and breeding workers can be helped to quickly screen individuals with excellent ethylene-sensitive exfoliation character in a seed or seedling stage. According to the method, the breeding efficiency is improved, the breeding period is shortened, the prediction accuracy is enhanced, the hereditary stability is improved, and the economic benefit is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular breeding, and in particular to a whole-genome SNP molecular marker combination for ethylene-sensitive shedding of rose and an application thereof. Background Art

[0002] Roses, a popular ornamental plant worldwide, hold a pivotal position in the horticulture and floriculture industries. Rose breeding has long been a hot topic in horticultural research. Ethylene-sensitive shedding, a key trait affecting the bloom lifespan and shelf life of cut roses, directly impacts the economic value and market competitiveness of roses. In-depth research on this trait and its application in breeding practice is of extraordinary significance.

[0003] Traditional breeding methods rely mainly on field phenotypic observations, which have many limitations. On the one hand, this method is inefficient. Breeders need to spend a lot of time and energy on field observations and records, and can only evaluate trait performance when the plants grow to a specific stage. The entire cycle is long, and it often takes several years or even longer to screen individuals with excellent traits. On the other hand, traditional breeding is greatly affected by environmental factors. Slight changes in environmental conditions such as temperature, humidity, and light may interfere with phenotypic observation results, resulting in inaccurate data, increasing the difficulty and uncertainty of screening excellent varieties. Furthermore, traditional breeding is difficult to achieve accurate predictions. Due to limited understanding of the molecular mechanism of rose ethylene-sensitive shedding traits, it is impossible to accurately grasp the control rules of genes on traits. Screening individuals that meet the target traits from complex breeding materials is like looking for a needle in a haystack, and the breeding direction is prone to deviations.

[0004] With the rapid development of molecular biology, molecular marker technology has emerged and gradually gained prominence. As a third-generation molecular marker, SNP markers have become a mainstream tool in molecular breeding due to their numerous advantages, including high detection throughput, dense distribution, and strong specificity. Their application in rose breeding has brought hope for overcoming traditional breeding challenges. In contrast, earlier molecular marker technologies, such as RFLP markers, were cumbersome to operate and their reliance on enzyme cleavage sites limited their application. RAPD markers, while simple to operate, lacked stability and were susceptible to experimental conditions. While SSR markers offered advantages such as rich polymorphism, they had a long development cycle and relatively high costs. The emergence of SNP markers has overcome these limitations. SNP markers are ubiquitous throughout the genome, enabling more accurate reflection of genetic variation within the genome, providing an efficient and precise tool for rose gene mapping and marker-assisted selection.

[0005] The emergence of genome-wide association analysis (GWAS) technology has revolutionized rose breeding. This technology enables researchers to construct predictive models of genotype and phenotype, enabling them to accurately predict future traits simply by testing the DNA of seeds or seedlings, without waiting for plants to mature. This fundamentally shortens the breeding cycle and significantly improves breeding efficiency. Through GWAS, gene loci closely associated with target traits can be quickly identified, laying the foundation for subsequent gene cloning and functional verification.

[0006] However, it's important to note that many gaps remain in the research on molecular markers for rose ethylene-sensitive shedding. The limited number of reported SNPs associated with ethylene-sensitive shedding makes it difficult to fully understand the genetic regulatory network underlying this complex trait, limiting the scope and effectiveness of molecular marker-assisted breeding. Furthermore, existing technologies still have significant room for improvement in accurately predicting genotypes and ensuring stable inheritance of desirable traits, failing to meet the stringent requirements of modern molecular breeding for efficiency, accuracy, and stability. Summary of the Invention

[0007] In order to solve the problems of the prior art, the present invention aims to provide a genome-wide SNP molecular marker combination for ethylene-sensitive shedding of rose and its application.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present application provides a genome-wide SNP molecular marker combination for ethylene-sensitive shedding in rose.

[0010] In a second aspect, the present application provides a predictive probe for ethylene-sensitive shedding of roses.

[0011] In a third aspect, the present application provides a chip for predicting ethylene-sensitive shedding of roses.

[0012] In a fourth aspect, the present application provides an application of a SNP molecular marker combination, probe or chip in the prediction of ethylene-sensitive shedding in roses.

[0013] In a fifth aspect, the present application provides a method for predicting ethylene-sensitive shedding of roses based on a combination of SNP molecular markers.

[0014] In a sixth aspect, the present application provides an application of a SNP site combination, probe, chip or prediction method in rose molecular breeding.

[0015] In a seventh aspect, the present application provides a rose molecular breeding method based on SNP markers.

[0016] The first aspect of the present application provides a genome-wide SNP molecular marker combination for ethylene-sensitive shedding in rose, the SNP molecular marker combination including 528 SNP sites distributed on 14 chromosomes of rose;

[0017] The specific information of the SNP site is represented by the structure of chromosome number_physical position_allele type. The SNP site contains a dominant allele that is positively and significantly associated with ethylene-sensitive shedding of rose. The specific site information is shown in Appendix 1 of the specification.

[0018] Furthermore, the SNP sites were determined by genome-wide association analysis of 358 rose resequencing materials and association screening using multiple software and models.

[0019] The second aspect of the present application provides a prediction probe for ethylene-sensitive shedding of roses. The probe is a liquid phase probe, and its sequence is designed according to the SNP site. It is used to hybridize with the rose genomic DNA and capture the DNA fragments in the region where the target SNP site is located so as to perform subsequent sequencing analysis to determine the genotype of the rose to be tested, thereby predicting its ethylene-sensitive shedding trait.

[0020] The third aspect of the present application provides a prediction chip for ethylene-sensitive shedding of roses, which includes probes for detecting the 528 SNP sites. By detecting the genotypes of these SNP sites in the genomic DNA of the rose samples to be tested and using a pre-constructed whole-genome selection model, accurate prediction of the ethylene-sensitive shedding trait of roses can be achieved.

[0021] The fourth aspect of the present application provides an application of a SNP molecular marker combination, probe or chip in the prediction of ethylene-sensitive shedding of roses, by extracting genomic DNA from rose plants and using SNP site combinations, probes or chips for detection.

[0022] A fifth aspect of the present application provides a method for predicting ethylene-sensitive shedding of roses based on a combination of SNP molecular markers, comprising the following steps:

[0023] (1) Extracting genomic DNA from the rose plants to be tested;

[0024] (2) constructing a library of the genomic DNA;

[0025] (3) using probes to perform hybridization capture on the DNA fragments in the library to enrich the DNA fragments containing the target SNP site;

[0026] (4) performing high-throughput sequencing on the captured DNA fragments;

[0027] (5) comparing the sequenced data with the rose reference genome to determine the genotype of the SNP site;

[0028] (6) The genotype data of the SNP site is input into the whole genome selection model to obtain the prediction results of the ethylene-sensitive shedding trait of the tested rose.

[0029] The sixth aspect of the present application provides an application of a SNP site combination, probe, chip or prediction method in rose molecular breeding, which is used for early, accurate and efficient prediction and screening of the ethylene-sensitive shedding traits of breeding materials during the rose breeding process, thereby shortening the breeding cycle, improving selection efficiency, and cultivating new rose varieties with excellent ethylene-sensitive shedding traits.

[0030] A seventh aspect of the present application provides a rose molecular breeding method based on SNP markers, comprising the following steps:

[0031] (1) Extracting rose genomic DNA;

[0032] (2) using the above-mentioned liquid-phase breeding chip to capture and enrich genomic DNA;

[0033] (3) high-throughput sequencing of the captured and enriched DNA;

[0034] (4) Analyze the sequencing data and detect the genotypes of the 528 SNP sites.

[0035] Beneficial effects: The method of the present invention breaks through the limitation of traditional breeding relying on field phenotypic screening, significantly shortens the breeding cycle, improves breeding efficiency, reduces breeding costs, and provides a revolutionary technical means for the selection and breeding of new rose varieties.

[0036] Compared with the prior art, the present invention has the following advantages:

[0037] (1) Shortening the breeding cycle: Traditional breeding methods rely on field phenotypic observations, requiring time-consuming trait assessments after the rose plants have grown to a certain stage. However, the present invention utilizes SNP molecular marker combinations, probes, or microarrays to rapidly detect genotypes associated with ethylene-sensitive shedding traits in roses at the seed or seedling stage, enabling early prediction and significantly shortening the breeding cycle.

[0038] (2) Efficient screening: Through high-throughput SNP detection technology, such as chip technology, a large number of rose breeding materials can be genotyped at the same time, and individuals carrying excellent genotypes can be quickly screened out, which improves the screening efficiency and enables breeders to process more breeding materials in a shorter time, thus accelerating the breeding process.

[0039] (3) Accurate genotyping: SNP molecular markers are highly specific and stable, accurately reflecting the genotype of individual roses at loci associated with ethylene-sensitive shedding. Using these SNP markers for testing can more accurately determine whether an individual carries superior genes, reducing misjudgments caused by factors such as phenotypic variation and environmental influences in traditional breeding and improving prediction accuracy.

[0040] (4) Whole-genome selection model assistance: Combining whole-genome selection models, such as the Bayes Ridge Regression (BRR) model, can fully utilize the genotypic information of SNP markers to establish a quantitative relationship between genotype and phenotype, further improving the prediction accuracy of ethylene-sensitive shedding traits. This model can comprehensively consider the micro-effects of multiple SNP markers, more comprehensively evaluate the genetic potential of individuals, and provide a more reliable basis for breeding decisions.

[0041] (5) Protecting superior genotypes: By systematically protecting SNPs associated with ethylene-sensitive shedding traits, we can prevent these key gene loci from mutating due to environmental stress or operational errors during the breeding process, thereby ensuring the stable inheritance of superior traits. This helps to stably pass on the selected superior gene combinations to future generations, improving the genetic stability of breeding.

[0042] (6) Reduced trait segregation: In traditional breeding, due to limited understanding of multi-gene interaction networks, it is difficult to precisely control trait inheritance, resulting in a relatively common phenomenon of trait segregation in offspring. However, the present invention, through precise SNP marker-assisted selection, can more specifically retain and accumulate genes related to the excellent ethylene-sensitive shedding trait, reduce the occurrence of unfavorable genotypes in offspring, reduce the risk of trait segregation, and enable more stable expression of excellent traits in offspring.

[0043] (7) Revealing the genetic basis: The 528 SNP markers screened in this study that are significantly associated with ethylene-sensitive abscission in roses and their distribution information provide important clues and entry points for further in-depth research on the genetic mechanism of ethylene-sensitive abscission in roses. Functional analysis of the genes containing these SNPs helps to reveal the molecular regulatory network of ethylene-sensitive abscission in roses, providing a new theoretical basis for understanding the genetic regulation of plant morphological development.

[0044] (8) Providing a research model: This SNP marker combination and its associated genome-wide selection model provide a method and approach that can be used as a reference for genetic research and breeding of ethylene-sensitive abscission in other plants. Other plant researchers can refer to the technical routes and methods of this invention to conduct research on ethylene-sensitive abscission traits in corresponding plants, thereby promoting the development of the entire field of plant molecular breeding.

[0045] (9) Reducing breeding costs: Although molecular technologies such as SNP detection require a certain amount of equipment and technical investment in the early stages, in the long run, by improving breeding efficiency and accuracy, it can significantly reduce breeding costs by reducing ineffective labor and resource waste in the breeding process. For example, it can reduce the cost of repeated hybridization and planting operations due to screening out individuals that do not meet the requirements, as well as the human, material and financial resources invested in the long breeding cycle.

[0046] (10) Increased added value: New rose varieties cultivated using the present invention that exhibit excellent ethylene-sensitive shedding traits are more in line with market demand and consumer preferences, and can enhance the market competitiveness and economic value of the products. For example, appropriate ethylene-sensitive shedding traits can extend the shelf life of cut roses, improve their quality and ornamental value, thereby increasing the added value of the products and bringing higher economic benefits to growers and related enterprises. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments or descriptions of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0048] Figure 1 This is the allele frequency diagram of ethylene-sensitive shedding in rose provided by the present invention.

[0049] Figure 2 This is a graph showing the loss rate of ethylene-sensitive shedding of roses provided by the present invention.

[0050] Figure 3 This is the heterozygosity diagram of ethylene-sensitive shedding of rose provided by the present invention.

[0051] Figure 4 The present invention provides a full chromosome marker distribution map of ethylene-sensitive shedding in rose.

[0052] Figure 5 This is a diagram showing the results of genome-wide selection evaluation of ethylene-sensitive shedding in roses provided by the present invention. DETAILED DESCRIPTION

[0053] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0054] In this application, the term "and / or" describes the relationship between associated objects, indicating that three possible relationships exist. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0055] In this application, "-one or more" means one or more, and "more than one" means two or more. "The following - one or more" or similar expressions refer to any combination of these items, including any combination of single items or plural items. For example, "a, b, or c - one or more", or "a, b, and c - one or more" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0056] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution. Some or all of the steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0057] The terms used in the embodiments of the present application are for the purpose of describing specific embodiments only and are not intended to limit the present application. The singular forms "a", "an", "the" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms unless the context clearly indicates otherwise.

[0058] The weights of the relevant components mentioned in the examples of this application may not only refer to the specific content of each component, but also represent the weight ratio between the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the examples of this application, it is within the scope disclosed in the examples of this application. Specifically, the mass described in the examples of this application may be a mass unit known in the chemical industry, such as μg, mg, g, kg, etc.

[0059] The terms "first" and "second" are used solely for descriptive purposes to distinguish objects, such as substances, from one another and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the technical features being referred to. For example, a first XX could also be referred to as a second XX, and similarly, a second XX could also be referred to as a first XX, without departing from the scope of the embodiments of this application. Thus, features defined as "first" or "second" may explicitly or implicitly include one or more of such features.

[0060] In a first aspect, the present invention provides a genome-wide SNP molecular marker combination for ethylene-sensitive shedding in rose, the SNP molecular marker combination comprising 528 SNP sites distributed on 14 chromosomes of rose;

[0061] The specific information of the SNP site is represented by the structure of chromosome number_physical position_allele type. The SNP site contains a dominant allele that is positively and significantly associated with ethylene-sensitive shedding of rose. The specific site information is shown in Table 1.

[0062] In some embodiments, the SNP sites are determined by performing genome-wide association analysis on 358 rose resequencing materials and screening with multiple software and model associations.

[0063] The second aspect of the embodiment of the present application provides a prediction probe for ethylene-sensitive shedding of roses. The probe is a liquid phase probe, and its sequence is designed according to the SNP site. It is used to hybridize with the rose genomic DNA and capture the DNA fragments in the region where the target SNP site is located so as to perform subsequent sequencing analysis to determine the genotype of the rose to be tested, thereby predicting its ethylene-sensitive shedding trait.

[0064] The third aspect of the embodiment of the present application provides a prediction chip for ethylene-sensitive shedding of roses. The prediction chip includes probes for detecting the 528 SNP sites. By detecting the genotypes of these SNP sites in the genomic DNA of the rose samples to be tested and using a pre-constructed whole-genome selection model, accurate prediction of the ethylene-sensitive shedding trait of roses is achieved.

[0065] The fourth aspect of the embodiments of the present application provides an application of a SNP molecular marker combination, probe or chip in the prediction of ethylene-sensitive shedding of roses, by extracting genomic DNA from rose plants and using SNP site combinations, probes or chips for detection.

[0066] A fifth aspect of the present application provides a method for predicting ethylene-sensitive shedding in roses based on a combination of SNP molecular markers, comprising the following steps:

[0067] (1) Extracting genomic DNA from the rose plant to be tested; (2) Constructing a library of the genomic DNA; (3) Using probes to hybridize and capture DNA fragments in the library to enrich DNA fragments containing target SNP sites; (4) Performing high-throughput sequencing on the captured DNA fragments; (5) Comparing and analyzing the sequenced data with the rose reference genome to determine the genotype of the SNP site; (6) Inputting the genotype data of the SNP site into the whole genome selection model to obtain the prediction result of the ethylene-sensitive shedding trait of the rose to be tested.

[0068] The sixth aspect of the embodiments of the present application provides an application of a SNP site combination, probe, chip or prediction method in rose molecular breeding, which is used for early, accurate and efficient prediction and screening of the ethylene-sensitive shedding traits of breeding materials during the rose breeding process, thereby shortening the breeding cycle, improving the selection efficiency, and cultivating new rose varieties with excellent ethylene-sensitive shedding traits.

[0069] A seventh aspect of the present invention provides a rose molecular breeding method based on SNP markers, comprising the following steps:

[0070] (1) Extracting rose genomic DNA;

[0071] (2) using the above-mentioned liquid-phase breeding chip to capture and enrich genomic DNA;

[0072] (3) high-throughput sequencing of the captured and enriched DNA;

[0073] (4) Analyze the sequencing data and detect the genotypes of the 528 SNP sites.

[0074] Example 1

[0075] The present invention provides a whole-genome SNP molecular marker combination for ethylene-sensitive shedding of rose, which includes 528 SNP sites distributed on 14 chromosomes of rose;

[0076] The specific information of the SNP site is represented by the structure of chromosome number_physical position_allele type. The SNP site contains a dominant allele that is positively and significantly associated with ethylene-sensitive shedding of rose. The specific site information is shown in Appendix 1 of the specification.

[0077] Table 1

[0078] Chr1A_20864114_G / T,Chr1B_14082864_G / A,Chr1B_20607409_G / A,Chr1B_20858301_T / C,Chr1B_42902690_G / A,Chr1B_46194211_C / T,Chr1B_46538140_A / G,Chr1B_46767907_A / G,Chr1B_46850397_T / A,Chr1B_46850403_A / G,Chr1B_46936761_C / T,Chr1B_46955389_T / C,Chr1B_47352354_A / T,Chr2A_64808336_A / G,Chr2A_68191644_A / G,Chr2A_80648862_C / T,Chr2A_80913132_G / T,Chr2A_81294110_C / G,Chr2A_82213890_C / T,Chr2A_83390588_T / A,Chr2A_83390597_C / A,Chr2A_84825533_G / A,Chr2A_85892821_G / T,Chr2A_86127102_T / A,Chr2B_1728679_G / A,Ch r2B_44800684_T / C,Chr2B_73789107_C / A,Chr2B_73790598_A / G,Chr2B_73790607_A / C,Chr2B_73790681_A / G,Chr2B_74829999_G / T,Chr2B_75405559_G / A,Chr2B_75405721_C / T,Chr2B_78443457_A / G,Chr2B_78443506_T / C,Chr2B_78727620_G / A,Chr2B_81426028_T / C,Chr2B_81695368_G / A,Chr2B_81695425_T / C,Chr2B_82045413_T / C,Chr2B_82045655_A / T,Chr2B_82054844_G / A,Chr2B_82057366_G / T,Chr2B_82068761_G / A,Chr2B_82081539_T / C,Chr2B_82235810_A / G,Chr2B_82292899_G / T,Chr2B_82366432_A / T,Chr2B_82513744_C / T,Chr2B_82667799_T / C,Chr2B_82668041_G / A,Chr2B_82843909_C / A,Chr2B_82848062_A / G,Chr2B_83085819_A / C,Chr3A_31845695_G / T,Chr3A_32889010_A / G,Chr3A_33140128_C / A,Chr3A_33353813_G / A,Chr3A_33356219_G / A,Chr3A_33356698_C / T,Chr3A_33440623_T / C,Chr3A_33440629_G / A,Chr3A_33440685_C / T,Chr3A_33440923_A / G,Chr3A_33440925_A / G,Chr3A_33444981_C / T,Chr3A_33479943_A / G,Chr3A_33479945_T / A,Chr3A_33479964_C / T,Chr3A_33525018_G / C,Chr3A_33613271_A / G,Chr3A_33639922_A / T,Chr3A_33680766_C / T,Chr3A_33708777_G / A,Chr3A_33708801_A / G,Chr3A_33708812_C / A,Chr3A_33708987_A / C,Chr3A_33717487_C / A,Chr3A_33732576_G / T,Chr3A_33771343_A / T,Chr3A_33772203_G / C,Chr3A_33774912_G / T,Chr3A_33775076_G / A,Chr3A_33808396_C / T,Chr3A_33808514_C / G,Chr3A_33811291_T / C,Chr3A_33835145_A / G,Chr3A_33836793_T / C,Chr3A_33877466_C / T,Chr3A_33877571_G / A,Chr3A_33877990_G / A,Chr3A_33878060_C / T,Chr3A_33878102_C / T,Chr3A_33878415_C / T,Chr3A_33878434_C / G,Chr3A_33879149_C / G,Chr3A_33879382_T / A,Chr3A_33879410_A / T,Chr3A_33879427_A / G,Chr3A_33880252_G / A,Chr3A_33880399_A / C,Chr3A_33880481_C / T,Chr3A_33880608_C / A,Chr3A_33880736_C / T,Chr3A_33880744_G / A,Chr3A_33880793_A / G,Chr3A_33881377_G / A,Chr3A_33881574_C / T,Chr3A_33885469_G / A,Chr3A_33889712_A / G,Chr3A_33919790_G / A,Chr3A_33934636_C / G,Chr3A_34050044_A / T,Chr3A_34050202_G / A,Chr3A_34050236_C / A,Chr3A_34050423_T / G,Chr3A_34050789_A / G,Chr3A_34051046_G / C,Chr3A_34051487_G / C,Chr3A_34051488_G / T,Chr3A_34051489_G / A,Chr3A_34051516_A / G,Chr3A_34051856_C / A,Chr3A_34052019_A / T,Chr3A_34052739_T / C,Chr3A_34055870_T / C,Chr3A_34055879_G / A,Chr3A_34060665_G / A,Chr3A_34067098_C / G,Chr3A_34068758_T / C,Chr3A_34068767_C / T,Chr3A_34068788_C / T,Chr3A_34068792_C / T,Chr3A_34068816_G / A,Chr3A_34068854_A / T,Chr3A_34068855_A / G,Chr3A_34088909_C / T,Chr3A_34088968_A / C,Chr3A_34094565_C / A,Chr3A_34094568_A / G,Chr3A_34097347_C / T,Chr3A_34133827_C / A,Chr3A_34133828_A / G,Chr3A_34133841_C / T,Chr3A_34193361_A / G,Chr3A_34193448_G / C,Chr3A_34301020_A / G,Chr3A_34343018_G / A,Chr3A_34392085_T / C,Chr3A_34392093_C / T,Chr3A_34392098_C / T,Chr3A_34392117_T / C,Chr3A_34392121_A / G,Chr3A_34392132_T / A,Chr3A_34392143_A / G,Chr3A_34392198_A / G,Chr3A_34392284_C / T,Chr3A_34392289_G / C,Chr3A_34392304_A / G,Chr3A_34401086_T / C,Chr3A_34401878_T / C,Chr3A_34403891_C / A,Chr3A_34404153_G / T,Chr3A_34404256_A / C,Chr3A_34404258_T / A,Chr3A_34404267_G / T,Chr3A_34404315_C / T,Chr3A_34404807_T / A,Chr3A_34404812_C / T,Chr3A_34404814_T / C,Chr3A_34405149_A / G,Chr3A_34405158_A / G,Chr3A_34406113_C / A,Chr3A_34406239_A / T,Chr3A_34409172_A / G,Chr3A_34409305_A / G,Chr3A_34409324_G / C,Chr3A_34409332_C / T,Chr3A_34409366_T / A,Chr3A_34409436_A / T,Chr3A_34443218_G / T,Chr3A_34455009_C / T,Chr3A_34455027_A / G,Chr3A_34455081_T / C,Chr3A_34455976_T / C,Chr3A_34456729_C / T,Chr3A_34457330_G / T,Chr3A_34457540_G / T,Chr3A_34470575_A / C,Chr3A_34470737_C / A,Chr3A_34470870_G / A,Chr3A_34482032_C / T,Chr3A_34546066_G / C,Chr3A_34546151_G / T,Chr3A_34576864_T / A,Chr3A_34582918_G / A,Chr3A_34582971_G / A,Chr3A_34814964_C / T,Chr3A_34825896_A / C,Chr3A_34825956_G / A,Chr3A_34826214_T / A,Chr3A_34827103_C / T,Chr3A_34827247_G / T,Chr3A_34827361_C / T,Chr3A_34827368_C / T,Chr3A_34827381_A / G,Chr3A_34832685_G / A,Chr3A_34832745_C / A,Chr3A_34832750_C / T,Chr3A_34875966_G / C,Chr3A_34886289_C / G,Chr3A_34895944_C / G,Chr3A_34897517_A / T,Chr3A_35649687_T / C,Chr3A_35650044_A / G,Chr3A_35653088_G / A,Chr3A_35653448_C / G,Chr3A_35653582_A / G,Chr3A_35653588_G / C,Chr3A_35653589_G / A,Chr3A_35653783_C / T,Chr3A_35657123_G / A,Chr3A_35673071_G / A,Chr3A_35687722_A / G,Chr3A_35690814_C / T,Chr3A_35725773_C / T,Chr3A_35751273_C / A,Chr3A_35768508_T / C,Chr3A_35801364_G / T,Chr3A_35801421_A / T,Chr3A_35858593_T / C,Chr3A_35859893_G / A,Chr3A_35861054_C / T,Chr3A_35868329_C / T,Chr3A_35886665_T / C,Chr3A_35898546_G / A,Chr3A_35900113_G / A,Chr3A_35927682_T / C,Chr3A_36148634_G / A,Chr3A_36148660_T / C,Chr3A_36148665_G / C,Chr3A_36148666_A / C,Chr3A_36148670_T / A,Chr3A_36148673_G / A,Chr3A_36152750_C / T,Chr3A_36435209_G / T,Chr3A_36435274_C / T,Chr3A_36435564_G / A,Chr3A_36435947_T / C,Chr3A_36436369_T / C,Chr3A_36436980_G / A,Chr3A_36437035_C / T,Chr3A_36465081_G / A,Chr3A_36711636_T / G,Chr3A_36712823_T / A,Chr3A_36712824_T / C,Chr3A_36716520_G / A,Chr3A_36717161_G / A,Chr3A_36723659_C / G,Chr3A_36723832_C / A,Chr3A_36723840_A / G,Chr3A_36724165_G / A,Chr3A_36733666_A / T,Chr3A_36733882_T / C,Chr3A_36734210_A / C,Chr3A_36807779_G / A,Chr3A_36807785_C / A,Chr3A_36808848_T / A,Chr3A_36809016_T / G,Chr3A_36809239_A / C,Chr3A_36809517_A / G,Chr3A_36810365_C / A,Chr3A_36812727_G / A,Chr3A_36812742_C / T,Chr3A_36813627_C / T,Chr3A_36829037_T / C,Chr3A_36829055_G / T,Chr3A_36829096_T / A,Chr3A_36829102_T / G,Chr3A_36829408_T / C,Chr3A_36835132_T / A,Chr3A_36837781_A / G,Chr3A_36838094_G / T,Chr3A_36838101_G / A,Chr3A_36838102_A / T,Chr3A_36838105_T / C,Chr3A_36838766_G / A,Chr3A_36842037_T / C,Chr3A_36906319_T / A,Chr3A_36906327_G / A,Chr3A_37183763_A / G,Chr3A_37183768_A / C,Chr3A_37568145_A / C,Chr3A_39108914_G / A,Chr3A_39108915_A / G,Chr3B_31254293_G / A,Chr3B_31328530_C / T,Chr3B_31328546_C / T,Chr3B_31329065_C / T,Chr3B_31329083_G / A,Chr3B_31329246_C / T,Chr3B_31329479_G / C,Chr3B_31329487_T / G,Chr3B_31865738_G / A,Chr3B_31866284_C / T,Chr3B_31869797_G / A,Chr3B_31890707_G / T,Chr3B_31890739_G / A,Chr3B_31890774_C / T,Chr3B_31890852_A / C,Chr3B_31926829_A / G,Chr3B_31931890_C / A,Chr3B_33823516_T / C,Chr3B_34212397_C / G,Chr3B_35954938_C / T,Chr4A_1870520_A / G,Chr4A_1870710_C / T,Chr4A_23256611_T / C,Chr4A_24214090_G / C,Chr4A_24214118_C / T,Chr4A_28137344_T / G,Chr4A_28158572_G / A,Chr4A_31947149_C / T,Chr4A_32447959_A / C,Chr4A_41544814_A / C,Chr4A_41605671_G / A,Chr4A_41607109_G / A,Chr4A_42054111_T / A,Chr4A_42054443_G / T,Chr4A_50982987_T / C,Chr5A_1445324_T / C,Chr5A_2240296_T / A,Chr5A_2771207_G / A,Chr5A_5122280_A / G,Chr5A_10357979_G / A,Chr5A_10521519_T / C,Chr5A_10660493_A / G,Chr5A_10660625_G / A,Chr5A_10660674_G / A,Chr5A_11049385_G / A,Chr5A_11050376_T / A,Chr5A_11050389_T / G,Chr5A_11052320_C / A,Chr5A_11250701_G / A,Chr5A_11873936_G / C,Chr5A_11887676_T / G,Chr5A_13027283_T / C,Chr5A_13798388_C / G,Chr5A_13887975_G / T,Chr5A_15119890_C / T,Chr5A_15119895_T / G,Chr5A_24300111_T / C,Chr5A_24300860_A / T,Chr5A_44076692_G / A,Chr5A_72938392_G / T,Chr5B_10537260_G / A,Chr5B_11191668_G / T,Chr5B_11380975_T / G,Chr5B_11381009_G / A,Chr5B_11384518_T / C,Chr5B_11708514_T / C,Chr5B_11709198_A / T,Chr5B_11711465_A / T,Chr5B_11711662_C / G,Chr5B_11712019_T / G,Chr5B_11712022_C / G,Chr5B_11712061_A / G,Chr5B_12421597_G / A,Chr5B_12823015_A / G,Chr5B_15650693_T / A,Chr5B_40359822_T / A,Chr5B_60801474_C / A,Chr5B_60801488_A / G,Chr5B_60801500_A / G,Chr5B_63134491_G / A,Chr5B_68795113_G / A,Chr6A_46839772_C / T,Chr6A_46927123_G / A,Chr6A_46927298_T / C,Chr6A_47042236_G / T,Chr6A_47045150_G / A,Chr6A_47377311_T / G,Chr6A_47568350_C / T,Chr6A_47568571_A / T,Chr6A_47569113_A / C,Chr6A_47712879_T / G,Chr6A_47721728_C / T,Chr6A_47753712_C / T,Chr6A_47753777_T / C,Chr6A_47754001_C / A,Chr6A_47754592_T / C,Chr6A_47754593_C / T,Chr6A_47754782_A / G,Chr6A_47832878_C / T,Chr6A_47832898_A / C,Chr6A_47833512_T / A,Chr6A_47833642_T / G,Chr6A_47833659_T / C,Chr6A_47836616_T / C,Chr6A_47840735_G / A,Chr6A_47843091_C / T,Chr6A_47843169_C / T,Chr6A_47843213_G / A,Chr6A_47843360_G / A,Chr6A_47843379_G / A,Chr6A_47843431_G / T,Chr6A_47843511_G / A,Chr6A_47952653_T / C,Chr6A_48000083_T / C,Chr6A_48080948_C / T,Chr6A_48087735_A / C,Chr6A_49665740_A / G,Chr6A_52992464_G / A,Chr6A_57841164_C / T,Chr6A_58003976_T / C,Chr6A_58063866_C / T,Chr6A_58911491_C / T,Chr6A_59026654_T / C,Chr6A_59026776_A / T,Chr6A_59026796_C / A,Chr6A_59034638_A / G,Chr6A_59034785_T / C,Chr6A_59035012_A / G,Chr6A_59056967_C / T,Chr6A_59093198_G / A,Chr6A_59093454_C / T,Chr6A_59093475_C / T,Chr6A_59096729_T / C,Chr6A_59119731_A / G,Chr6A_59252499_T / C,Chr6A_59306977_T / G,Chr6A_59447867_A / T,Chr6A_59447872_A / G,Chr6A_59447877_C / T,Chr6A_59447897_T / A,Chr6A_59447899_C / T,Chr6A_59447993_A / C,Chr6A_59448060_C / G,Chr6A_59502115_A / C,Chr6A_59659860_G / T,Chr6A_59735885_T / G,Chr6A_59792446_C / A,Chr6A_59858916_A / G,Chr6A_60022994_A / G,Chr6A_60042530_C / G,Chr6A_60076642_G / T,Chr6A_60112535_T / C,Chr6A_60117209_G / T,Chr6A_60208060_T / C,Chr6A_60270508_G / T,Chr6A_60270520_A / T,Chr6A_60527529_A / G,Chr6A_60527565_A / G,Chr6A_60527787_A / G,Chr6A_60527925_T / C,Chr6A_60528292_T / C,Chr6A_60534979_T / C,Chr6A_60535066_T / C,Chr6A_60543351_C / T,Chr6A_60567636_C / G,Chr6A_60576815_C / T,Chr6A_60704243_T / G,Chr6A_61343842_T / C,Chr6B_6994691_A / G,Chr6B_23377994_G / A,Chr6B_56883058_G / A,Chr7A_6375063_T / C,Chr7A_6544562_T / A,Chr7A_8393121_C / T,Chr7A_29148487_C / T,Chr7A_30350549_T / C,Chr7A_30401377_A / G,Chr7A_30496147_A / C,Chr7A_57656180_T / G,Chr7A_60303364_G / A,Chr7A_63467651_A / G,Chr7A_63467711_C / T,Chr7A_63467736_C / T,Chr7A_63467744_A / G,Chr7A_63467771_T / C,Chr7A_63467873_T / A,Chr7A_63467915_G / A, Chr7A_63467921_A / C,Chr7A_63467923_T / C,Chr7A_63468067_T / G,Chr7A_63468069_C / T,Chr7A_63468072_T / G,Chr7A_63468085_T / C,Chr7A_63468113_A / G,Chr7A_63468118_G / A,Chr7A_63468121_G / A,Chr7A_63468130_T / G,Chr7A_63468230_A / G,Chr7A_63468291_G / C,Chr7A_63468305_T / C, Chr7A_63468325_C / T,Chr7A_63468338_C / T,Chr7A_63468556_T / A,Chr7A_63468584_C / A,Chr7A_63468785_T / G,Chr7A_63468814_A / G,Chr7A_63468825_C / G,Chr7A_63469102_T / C,Chr7A_63469118_A / C,Chr7A_63469136_T / A,Chr7A_63469242_T / G,Chr7A_63469329_T / C,Chr7A_63469347_C / T, Chr7A_63469355_C / T,Chr7A_63469398_T / A,Chr7A_63469400_C / T,Chr7A_63469414_C / T,Chr7A_63469504_C / T,Chr7A_63469871_G / A,Chr7A_63471571_G / T,Chr7A_63471977_C / T,Chr7A_63473025_A / C,Chr7A_68661193_T / C,Chr7A_69309715_A / C,Chr7A_69316691_A / G,Chr7A_69323092_C / A,Chr7B_9431658_T / A,Chr7B_44115932_A / G,Chr7B_44115961_G / T,Chr7B_4462 1419_C / A,Chr7B_44621726_C / T,Chr7B_46177675_G / A,Chr7B_50827758_T / C,

[0079] The SNP sites were determined through genome-wide association analysis of 358 rose resequencing materials and association screening using multiple software and models.

[0080] Example 2

[0081] The present invention discloses a prediction probe for ethylene-sensitive shedding of roses. The probe is a liquid phase probe, the sequence of which is designed according to the SNP site, and is used to hybridize with rose genomic DNA to capture DNA fragments in the region where the target SNP site is located, so as to perform subsequent sequencing analysis to determine the genotype of the rose to be tested, thereby predicting its ethylene-sensitive shedding trait.

[0082] Example 3

[0083] The present invention provides a prediction chip for ethylene-sensitive shedding of roses, which includes probes for detecting the 528 SNP sites. By detecting the genotypes of these SNP sites in the genomic DNA of the rose samples to be tested and utilizing a pre-constructed whole-genome selection model, accurate prediction of the ethylene-sensitive shedding trait of the roses can be achieved.

[0084] Example 4

[0085] The invention discloses an application of a SNP molecular marker combination, a probe or a chip in the prediction of ethylene-sensitive shedding of roses, which involves extracting genomic DNA from rose plants and performing detection using the SNP site combination, the probe or the chip.

[0086] Example 5

[0087] A method for predicting ethylene-sensitive shedding of roses based on a combination of SNP molecular markers of the present invention comprises the following steps:

[0088] (1) Extracting genomic DNA from the rose plants to be tested;

[0089] (2) constructing a library of the genomic DNA;

[0090] (3) using probes to perform hybridization capture on the DNA fragments in the library to enrich the DNA fragments containing the target SNP site;

[0091] (4) performing high-throughput sequencing on the captured DNA fragments;

[0092] (5) Compare the sequencing data with the rose reference genome to determine the genotype of the SNP site;

[0093] (6) The genotype data of the SNP site is input into the whole genome selection model to obtain the prediction results of the ethylene-sensitive shedding trait of the tested rose.

[0094] Example 6

[0095] The application of a SNP site combination, probe, chip or prediction method of the present invention in rose molecular breeding is used to perform early, accurate and efficient prediction and screening of the ethylene-sensitive shedding trait of breeding materials during the rose breeding process, thereby shortening the breeding cycle, improving selection efficiency, and cultivating new rose varieties with excellent ethylene-sensitive shedding traits.

[0096] Example 7

[0097] The present invention provides a rose molecular breeding method based on SNP markers, comprising the following steps:

[0098] (1) Extracting rose genomic DNA;

[0099] (2) using the above-mentioned liquid-phase breeding chip to capture and enrich genomic DNA;

[0100] (3) high-throughput sequencing of the captured and enriched DNA;

[0101] (4) Analyze the sequencing data and detect the genotypes of the 528 SNP sites.

[0102] Example 8

[0103] Analyze the genome-wide association analysis of ethylene-sensitive shedding from 358 rose genome resequencing data, including the following process:

[0104] (1) Genomic DNA from 358 rose leaf tissues was extracted using the magnetic bead method, and a whole-genome resequencing library was constructed. DNA fragments that met the sequencing requirements were screened by magnetic beads, and sequenced using BGI DNBSEQ-T7 after qbit quantification.

[0105] (2) The second generation sequencing data in fq.gz format obtained in step (1) were aligned with the rose R. hybrida'Samantha' genome (v2) using bwa software

[0106] The genome was analyzed using the GATK software (https: / / figshare.com / articles / dataset / _i_R_hybrida_i_Samantha_genome_v2_ / 28738781?file=53454104). SNP variation data were analyzed using GATK software, ultimately identifying 115,219,112 high-quality SNP sites.

[0107] (3) The SNP sites selected in step (2) are retained, and the SNP sites with allele frequencies greater than 0.01 and deletion rates less than 0.2 are retained. Figure 2 ), and obtained 35335433 high-quality SNP sites ( Figure 1 ).

[0108] (4) Collection of agronomic traits of ethylene-sensitive shedding: Select flower branches that are healthy, free of pests and diseases, and have the same development level, and conduct experiments in a standardized environment. Insert the flower branches vertically into the container, control the depth to be 10-15 cm, and change the vase liquid every 2-3 days. Use Guoguang ethephon solution (1 mL, diluted 2000 times) as the vase liquid. Ethylene sensitivity is defined as the time from the time the flower branch is inserted into the vase liquid to the time the flower falls off in days. Collect 3 repeated ethylene-sensitive shedding data, calculate the average value and BLUP value, and a total of 5 data are used for GWAS association analysis.

[0109] (5) Six models were used to correlate ethylene-sensitive shedding with the agronomic trait using four software programs: emmax, gemma, rmvp (FARMCPU model, GLM model, MLM model), and tassel (GLM model). The p-value threshold was set at 1E-4, and 268,779 SNP markers were consistently associated. After deduplication of the association results from different software models, 134,868 SNP loci were obtained.

[0110] (6) Probe evaluation was performed on the SNPs screened in step (5). Screening principles: there is no InDel marker within 50 bp upstream and downstream of the SNP site; there is only one copy number in the entire genome for each 100 bp upstream and downstream sequence; the GC content is 40-60%; there are no short fragment repeats in the sequence; there is no N base in the sequence, and finally 5710 SNP sites remain ( Figure 3 ).

[0111] (7) The SNP sites selected in step (6) were sorted according to the number of software and model associations and the association results, and the 2001 SNP markers with the largest number of software associations and significant association values ​​were selected. Figure 4 ).

[0112] (8) The SNP sites selected in step (7) were used to evaluate the prediction accuracy of ethylene-sensitive abscission using 13 whole-genome selection models, including LightGBM (lgb), Random Forest (RF), SVR (svr), Lasso Regression (Lasso), Ridge Regression (Ridge), rrBLUP (rrblup), Reproducing Kernel Hilbert Space (RKHS), Bayes Ridge Regression (BRR), BayesianLasso (BL), BayesC, BayesB, BayesA, and GBLUP. The genotypes and agronomic traits of ethylene-sensitive abscission of 348 materials were used to evaluate the prediction accuracy of ethylene-sensitive abscission. The results showed that Ridge had the lowest prediction accuracy, while Bayes Ridge Regression (BRR) had the highest prediction accuracy. The overall prediction accuracy was greater than 0.639, and the prediction accuracy was good ( Figure 5 ).

[0113] (9) The SNP sites screened in step (7) are synthesized into probe sequences for capture sequencing.

[0114] Example 9

[0115] Method for detecting rose using the liquid phase probe corresponding to the 528 SNPs described in Example 8

[0116] Genomic DNA Extraction: Rose leaves were collected, stored in ice packs, and promptly transported back to the laboratory. Total DNA was extracted using a magnetic bead method. Genomic DNA Library Construction: Genomic DNA was fragmented (200-300 bp), end-repaired, and ligated with adapters for pre-PCR amplification of the library.

[0117] Hybridization of the probe with the target region: The prepared SNP probe is hybridized with the Pre-PCR library using an elution buffer of 0.1×SSC / 0.1% SDS. The streptavidin-modified probe is then used to capture the complementary DNA library. The target DNA library is enriched, and the remaining library DNA is eluted to form the captured Pre-PCR library. Hybridization conditions: The streptavidin-labeled probe is hybridized with the target library at 50°C for 16 hours. The target library is enriched by binding biotin-labeled magnetic beads to the streptavidin-labeled probe at room temperature for 30 minutes. The non-target region library is rinsed with wash buffer, and then eluted with nuclease-free water.

[0118] After magnetic bead purification, the DNA was amplified and enriched to the required DNA concentration for high-throughput sequencing, and then subjected to high-throughput sequencing. High-throughput sequencing: The library obtained by capture and amplification in step (4) was subjected to high-throughput sequencing using the MGI DNBSEQ-T7 sequencing platform to obtain the sequencing results of the genomic DNA, and the obtained data was subjected to basic cleaning processing. Liquid phase probe capture efficiency evaluation: The cleaned sequencing data was aligned to the rose (R. hybrida 'Samantha') reference sequence using bwa software to evaluate its capture efficiency.

[0119] Test Example 1

[0120] Evaluation of liquid probe capture efficiency for 12 rose varieties

[0121] Experimental Materials: Twelve modern rose varieties with significant genetic differences were selected (see Table 2 for details). Fresh leaves were collected, immediately snap-frozen in liquid nitrogen, and stored at -80°C. Genomic DNA from each variety was extracted using the CTAB method (cetyltrimethylammonium bromide). DNA purity (OD260 / 280 = 1.8-2.0) and concentration (≥50 ng / μL) were confirmed by agarose gel electrophoresis and Nanodrop analysis. Sample numbers are shown in Table 2.

[0122] Table 2

[0123] serial number Rose varieties serial number Rose varieties Test1 Palio Test2 gold medal Test3 Moon Goddess Test4 Ruby Star Test5 Meilang lipstick Test6 Hongruisheng Test7 Lavender Test8 Angela Test9 Golden Lotus Test10 Holiday Princess Test11 Fragrant Joy Test12 Deep Blue Night

[0124] Variety Representativeness: The selected varieties cover six color families (red, yellow, white, pink, purple, and orange) and major commercial types (hybrid tea roses and floriferous roses). For example, red roses are represented by Paleo (Test 1) and Lipstick (Test 5); purple roses are represented by Lavender (Test 7) ​​and Deep Blue Night (Test 12); and yellow roses are represented by Gold Medal (Test 2) and Golden Lotus (Test 9). This design ensures universal validation of the probes across diverse genetic backgrounds.

[0125] Capture efficiency evaluation

[0126] Based on the evaluation results of 12 monthly materials, the proportion of 528 SNPs that can be detected ranges from 97.75% to 98.75%, with a coverage depth of 391.08-531.36X. The overall capture efficiency is good and can be used for subsequent SNP detection and whole-genome selection evaluation of other samples. Table 3 is a statistical table of the detection rate of capture sequencing samples of the present invention. The liquid probe library designed by the present invention (containing 528 target SNP sites) was used to perform hybridization capture on the samples shown in Table 2, and the Illumina NovaSeq 6000 platform performed 150bp double-end sequencing. The key indicators were calculated through bioinformatics processes (including Trimmomatic quality control, BWA alignment, and GATK variation detection): Call Rate: the proportion of target SNPs that are effectively detected; Coverage Depth: the average sequencing depth of the target area.

[0127] Table 3

[0128]

[0129]

[0130] Data analysis showed that: (1) the detection rate of all samples was ≥97.75% (mean 98.35% ± 0.31%), indicating that the number of effective detections was ≥516 out of 528 SNP sites; (2) the coverage depth ranged from 391.08X to 531.36X (mean 456.25X ± 47.82X), far exceeding the minimum depth threshold for SNP typing (≥30X); (3) key variety data: Xiang Huanxi (Test 11): had the highest detection rate (98.75%); Shenlan Zhiye (Test 12): had the highest coverage depth (531.36X). (4) capture efficiency stability: the detection rate coefficient of variation (CV) was only 0.32%, demonstrating the excellent applicability of the probe across varieties.

[0131] Technical Effects: The liquid-phase probe system described in this invention achieves: ① a target SNP capture efficiency ≥97.75%, meeting the typing accuracy requirement of genome-wide selection (GS) (>95%); ② ultra-high coverage depth (average 456.25X), effectively mitigating the risk of allele dropout; and ③ stable performance across 12 diverse cultivars (detection rate fluctuation range ≤1%), demonstrating the broad applicability of the probe design. Conclusion: This liquid-phase probe system can be effectively applied to rose germplasm SNP typing, population genetic analysis, and genome-wide selection breeding.

[0132] The basic principles, main features and advantages of the present invention are shown and described above. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and improvements may be made to the present invention without departing from the spirit and scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims, the description and their equivalents.

Claims

1. A genome-wide SNP molecular marker combination for ethylene-sensitive shedding in rose, characterized by: The SNP molecular marker combination includes 528 SNP sites distributed on 14 chromosomes of rose; The specific information of the SNP site is represented by the structure of chromosome number_physical position_allele type. The SNP site contains a dominant allele that is positively and significantly associated with ethylene-sensitive shedding of rose. The specific site information is shown in Appendix 1 of the specification.

2. The genome-wide SNP molecular marker combination for ethylene-sensitive shedding of rose according to claim 1, characterized in that: The SNP sites were determined by performing genome-wide association analysis on 358 rose resequencing materials and screening with multiple software and model associations.

3. A probe for predicting ethylene-sensitive shedding of rose, characterized by: The probe is a liquid phase probe, the sequence of which is designed according to the SNP site, and is used to hybridize with rose genomic DNA to capture DNA fragments in the region where the target SNP site is located, so as to perform subsequent sequencing analysis to determine the genotype of the rose to be tested, thereby predicting its ethylene-sensitive shedding trait.

4. A chip for predicting ethylene-sensitive shedding of roses, characterized by: The prediction chip includes probes for detecting the 528 SNP sites. By detecting the genotypes of these SNP sites in the genomic DNA of the rose sample to be tested and using a pre-constructed whole-genome selection model, accurate prediction of the ethylene-sensitive shedding trait of the rose is achieved.

5. Use of the SNP molecular marker combination according to claim 1, the probe according to claim 3, or the chip according to claim 4 in predicting ethylene-sensitive shedding of roses, characterized in that: The genomic DNA of the rose plant is extracted and detected using the SNP site combination, probe or chip.

6. A method for predicting ethylene-sensitive shedding of rose based on the SNP molecular marker combination according to claim 1, characterized in that The steps include: (1) Extracting genomic DNA from the rose plants to be tested; (2) constructing a library of the genomic DNA; (3) using the probe described in claim 3 to perform hybridization capture on the DNA fragments in the library to enrich the DNA fragments containing the target SNP site; (4) performing high-throughput sequencing on the captured DNA fragments; (5) comparing the sequenced data with the rose reference genome to determine the genotype of the SNP site; (6) Inputting the genotype data of the SNP site into the whole genome selection model described in claim 4 to obtain the prediction result of the ethylene-sensitive shedding trait of the tested rose.

7. Use of the SNP locus combination, probe, chip or prediction method according to claim 1 in rose molecular breeding, characterized in that: It is used to make early, accurate and efficient prediction and screening of the ethylene-sensitive shedding traits of breeding materials during the rose breeding process, thereby shortening the breeding cycle, improving selection efficiency, and cultivating new rose varieties with excellent ethylene-sensitive shedding traits.

8. A rose molecular breeding method based on SNP markers, characterized in that The steps include: (1) Extracting rose genomic DNA; (2) using the above-mentioned liquid-phase breeding chip to capture and enrich genomic DNA; (3) high-throughput sequencing of the captured and enriched DNA; (4) Analyze the sequencing data and detect the genotypes of the 528 SNP sites.

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