Rosa chinensis bottle longevity whole genome snp molecular marker combination and application thereof

Abstract

The application discloses a Rosa chinensis bottle insertion life whole genome SNP molecular marker combination and application thereof. The SNP site combination comprises 1679 SNP markers significantly associated with the bottle insertion life of Rosa chinensis and is uniformly distributed on 14 chromosomes of the Rosa chinensis. The SNP sites are screened through whole genome association analysis, and a whole genome selection model constructed by using the SNP sites can accurately predict the bottle insertion life of the Rosa chinensis, and the prediction accuracy can be above 0.710. The application also relates to specific probes designed for the SNP sites and a liquid breeding chip comprising the probes, and the liquid breeding chip can be used for rapidly and efficiently screening out individuals with excellent bottle insertion life traits at the seed or seedling stage of the Rosa chinensis, so that the breeding cycle is significantly shortened, the breeding efficiency is improved, and powerful technical support is provided for molecular breeding and variety improvement of the Rosa chinensis.

Description

Technical Field

[0001] This invention relates to the field of molecular breeding technology, and in particular to a genome-wide SNP molecular marker combination for vase life of roses and its application. Background Technology

[0002] As an important ornamental plant, the vase life of roses is a core indicator determining their ornamental and economic value, and is of great significance to the development of the rose industry. Traditional breeding methods rely on field phenotypic observation, which suffers from problems such as heavy workload, lengthy cycle, and low efficiency. Moreover, during the breeding process, the genotypes that determine key economic traits such as vase life are easily lost due to recombination or mutation, limiting the improvement of the efficiency of targeted breeding.

[0003] In recent years, SNP molecular marker technology has been widely applied, and genome-wide selection breeding technology has developed rapidly. This technology, by constructing predictive models of genotype and agronomic traits, can achieve efficient screening of materials at an early stage, breaking the limitations of traditional breeding that relies excessively on field phenotypic screening. However, existing gene prediction models are unable to grasp the complex genetic regulatory mechanisms behind the vase life of roses, with a prediction accuracy of less than 30%, which is insufficient to meet the urgent needs of the modern rose industry for improving traits such as vase life. Traditional breeding methods require plants to grow to a specific stage before trait evaluation can be carried out, which is time-consuming and inefficient.

[0004] Therefore, there is an urgent need to build a genotype protection technology system and develop accurate and efficient SNP marker combinations for predicting the vase life of roses and their applications. Summary of the Invention

[0005] To address the problems of existing technologies, the purpose of this invention is to provide a genome-wide SNP molecular marker combinatorial model for rose vase life and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In the first aspect, this application provides a genome-wide SNP molecular marker combination for the vase life of roses.

[0008] Secondly, this application provides a probe for predicting the vase life of roses.

[0009] Thirdly, this application provides a chip for predicting the vase life of roses.

[0010] Fourthly, this application provides an application of SNP molecular marker combinations, probes, or chips in predicting the vase life of roses.

[0011] Fifthly, this application provides a method for predicting the vase life of roses based on SNP molecular marker combinations.

[0012] Sixthly, this application provides an application of SNP site combinations, probes, chips, or prediction methods in the molecular breeding of roses.

[0013] Seventhly, this application provides a molecular breeding method for roses based on SNP markers.

[0014] The first aspect of this application provides a whole-genome SNP molecular marker combination for the vase life of a rose, the SNP molecular marker combination including 1679 SNP loci distributed on the 14 chromosomes of the rose;

[0015] The specific information of the SNP loci is represented in the structure of chromosome number_physical location_allele. The SNP loci contain dominant alleles that are positively and significantly associated with the vase life of roses. The specific locus information is shown in Table 1 of the specification.

[0016] Furthermore, the SNP sites were determined through genome-wide association analysis of 358 rose resequencing materials, and were screened using multiple software and models.

[0017] The second aspect of this application provides a probe for predicting the vase life of roses. The probe is a liquid-phase probe whose sequence is designed based on the SNP site. It is used to hybridize with the genomic DNA of roses 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 vase life trait.

[0018] A third aspect of this application provides a prediction chip for the vase life of roses. The prediction chip includes probes for detecting the 1679 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, the accurate prediction of the vase life trait of roses can be achieved.

[0019] The fourth aspect of this application provides an application of SNP molecular marker combinations, probes, or chips in predicting the vase life of roses, by extracting genomic DNA from rose plants and using the SNP site combinations, probes, or chips for detection.

[0020] The fifth aspect of this application provides a method for predicting the vase life of roses based on SNP molecular marker combinations, including the following steps:

[0021] (1) Extract genomic DNA from the rose plants to be tested;

[0022] (2) Constructing a library of genomic DNA;

[0023] (3) Use probes to hybridize and capture DNA fragments in the library to enrich DNA fragments containing target SNP sites;

[0024] (4) Perform high-throughput sequencing on the captured DNA fragments;

[0025] (5) Compare and analyze the sequencing data with the rose reference genome to determine the genotype of the SNP site;

[0026] (6) Input the genotype data of the SNP loci into the whole genome selection model to obtain the prediction results of the vase life trait of the rose to be tested.

[0027] The sixth aspect of this application provides an application of SNP site combinations, probes, chips, or prediction methods in rose molecular breeding, which is used to predict and screen the vase life trait of breeding materials in an early, accurate, and efficient manner during the rose breeding process, thereby shortening the breeding cycle, improving selection efficiency, and cultivating new rose varieties with excellent vase life traits.

[0028] Furthermore, a genome-wide selection model constructed based on SNP site combinations can predict the vase life of roses with a prediction accuracy greater than 0.710.

[0029] The seventh aspect of this application provides a molecular breeding method for roses based on SNP markers, comprising the following steps:

[0030] (1) Extracting genomic DNA from roses;

[0031] (2) Use the above-mentioned liquid phase breeding chip to capture and enrich genomic DNA;

[0032] (3) Perform high-throughput sequencing on the captured and enriched DNA;

[0033] (4) Analyze the sequencing data and detect the genotypes of the 1679 SNP sites.

[0034] Beneficial effects: By using the selected SNP marker combinations and corresponding detection technologies, this invention can accurately predict the vase life traits of roses at the seed or seedling stage, without waiting for the plants to mature, which greatly shortens the breeding cycle, improves breeding efficiency, and accelerates the breeding process of new rose varieties.

[0035] Compared with the prior art, the present invention has the following advantages:

[0036] (1) Traditional gene prediction models are difficult to effectively control the complex genetic regulation mechanism of rose vase life, with a prediction accuracy of less than 30%. This invention successfully screened 1679 SNP markers that are significantly associated with rose vase life through genome-wide association analysis and constructed a genome-wide selection model with a prediction accuracy of over 0.710, which significantly improves the accuracy of rose vase life prediction and provides a more reliable screening basis for breeding work.

[0037] (2) This invention provides an in-depth analysis of the distribution characteristics and functional properties of SNP markers related to the vase life of roses, providing researchers with rich data resources and scientific and detailed theoretical basis, which helps to further explore the mechanism of action of related genes, clarify the potential targets for subsequent gene editing or genetic improvement work, greatly promote the development of rose molecular breeding technology, and inject new vitality and impetus into the variety improvement and market competitiveness enhancement of the rose industry.

[0038] (3) The liquid-phase breeding chip technology developed in this invention can quickly and accurately detect SNP sites that are significantly associated with key traits such as vase life. It has the characteristics of high throughput and high accuracy, effectively reducing costs and improving the efficiency and reliability of breeding material screening. It provides strong technical support for the targeted breeding and variety improvement of ornamental plants and has good application prospects and promotion value.

[0039] (4) The SNP marker combination and related technologies of the present invention can be used to construct a rose genotype protection technology system, which helps to maintain the excellent characteristics of rose varieties and prevent the loss or loss of function of genotypes that determine key economic traits such as vase life due to gene recombination or mutation in large-scale breeding practices, thus providing technical support for the stable inheritance and continuous improvement of rose varieties. Attached Figure Description

[0040] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0041] Figure 1 Allelic frequency map of rose vase life provided by the present invention.

[0042] Figure 2 The missing rate diagram of the vase life of roses provided by this invention.

[0043] Figure 3 The heterozygosity diagram of the vase life of roses provided by the present invention.

[0044] Figure 4 The whole chromosome marker distribution map of the vase life of roses provided by this invention.

[0045] Figure 5 The diagram shows the genome-wide selection evaluation results of the vase life of roses provided by this invention. Detailed Implementation

[0046] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0047] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0048] In this application, "-one less" means one or more, and "more than" means two or more. "-one less item (item) below" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "-one less item (item) in a, b, or c", or "-one less item (item) in a, b, and c", 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.

[0049] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may 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 this application.

[0050] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0051] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass described in the embodiments of this application can be a mass unit known in the chemical industry, such as μg, mg, g, or kg.

[0052] The first aspect of this application provides a whole genome SNP molecular marker combination for the vase life of a rose, the SNP molecular marker combination including 1679 SNP loci distributed on the 14 chromosomes of the rose;

[0053] The specific information of the SNP loci is represented in the structure of chromosome number_physical location_allele. The SNP loci contain dominant alleles that are positively and significantly associated with the vase life of roses. The specific locus information is shown in Table 1.

[0054] In some embodiments, SNP sites were determined by genome-wide association analysis of 358 rose resequencing materials, followed by association screening using multiple software and models.

[0055] The second aspect of this application provides a probe for predicting the vase life of roses. The probe is a liquid-phase probe whose sequence is designed based on the SNP site. It is used to hybridize with the genomic DNA of roses 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 vase life trait.

[0056] A third aspect of this application provides a prediction chip for the vase life of roses. The prediction chip includes probes for detecting the 1679 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, the accurate prediction of the vase life trait of roses can be achieved.

[0057] The fourth aspect of this application provides an application of SNP molecular marker combinations, probes, or chips in predicting the vase life of roses, by extracting genomic DNA from rose plants and using the SNP site combinations, probes, or chips for detection.

[0058] The fifth aspect of this application provides a method for predicting the vase life of roses based on SNP molecular marker combinations, including the following steps:

[0059] (1) Extract genomic DNA from the rose plants to be tested;

[0060] (2) Constructing a library of genomic DNA;

[0061] (3) Use probes to hybridize and capture DNA fragments in the library to enrich DNA fragments containing target SNP sites;

[0062] (4) Perform high-throughput sequencing on the captured DNA fragments;

[0063] (5) Compare and analyze the sequencing data with the rose reference genome to determine the genotype of the SNP site;

[0064] (6) The genotype data of SNP loci are input into the whole-genome selection model to obtain the prediction results of the vase life trait of the rose to be tested. In some embodiments, the whole-genome selection model constructed based on the combination of SNP loci can predict the vase life of roses with a prediction accuracy greater than 0.710. The model is implemented by integrating 13 types of algorithms, including: machine learning models: LightGBM, Random Forest, SVR; penalized regression models: Lasso, Ridge, rrBLUP; Bayesian models: BRR, BL, BayesC / B / B / A; and hybrid models: RKHS, GBLUP. Among them, Bayes Ridge Regression (BRR) is the optimal model.

[0065] The sixth aspect of this application provides an application of SNP site combinations, probes, chips, or prediction methods in rose molecular breeding, which is used to predict and screen the vase life trait of breeding materials in an early, accurate, and efficient manner during the rose breeding process, thereby shortening the breeding cycle, improving selection efficiency, and cultivating new rose varieties with excellent vase life traits.

[0066] In some embodiments, a genome-wide selection model based on SNP site combinations can predict the vase life of roses with a prediction accuracy greater than 0.710.

[0067] The seventh aspect of this application provides a method for molecular breeding of roses based on SNP markers, comprising the following steps:

[0068] (1) Extracting genomic DNA from roses;

[0069] (2) Use the above-mentioned liquid phase breeding chip to capture and enrich genomic DNA;

[0070] (3) Perform high-throughput sequencing on the captured and enriched DNA;

[0071] (4) Analyze the sequencing data and detect the genotypes of the 1679 SNP sites.

[0072] Example 1

[0073] The present invention provides a genome-wide SNP molecular marker combinatorial analysis of rose vase life, comprising 1679 SNP loci distributed on the 14 chromosomes of the rose.

[0074] The specific information of the SNP loci is represented in the structure of chromosome number_physical location_allele. The SNP loci contain dominant alleles that are positively and significantly associated with the vase life of roses. The specific locus information is shown in Table 1.

[0075] Table 1

[0076] Chr1A_4813520_A / G,Chr1A_4813537_T / C,Chr1A_30834011_C / T,Chr1A_32221286_C / T,Chr1A_32223961_G / A,Chr1A_33011346_C / T,Chr1A_44091992_A / C,Chr1A_44092015_T / C,Chr1A_44092019_T / G,Chr1A_44092068_C / T,Chr1A_45921074_G / A,Chr1A_46150241_C / T,Chr1A_46225710_C / T,Chr1A_53233599_G / A,Chr1A_57148115_A / T,Chr1A_57177061_T / C,Chr1A_57177067_A / T,Chr1A_57198742_C / G,Chr1A_57420833_T / C,Chr1A_57555262_C / T,Chr1A_57555276_T / A,Chr1A_57556169_A / T,Chr1A_57629705_G / T,Chr1A_66409844_G / A,Chr1B_2660966_T / A,Chr1B_4958719_G / C,Chr1B_4958724_A / C,Chr1B_5726434_T / C,Chr1B_10905523_G / A,Chr1B_30541369_G / T,Chr1B_42202981_C / T,Chr1B_42480174_T / C,Chr1B_42775273_C / T,Chr1B_42832224_A / G,Chr1B_42875461_C / G,Chr1B_42875469_C / T,Chr1B_42948251_A / G,Chr1B_42948257_T / C,Chr1B_42948335_T / C,Chr1B_42948342_A / G,Chr1B_42948362_A / G,Chr1B_42948374_T / A,Chr1B_42948391_G / A,Chr1B_42948417_C / A,Chr1B_42948418_G / A,Chr1B_42948427_T / C,Chr1B_42948444_C / G,Chr1B_42987522_A / G,Chr1B_43030552_C / A,Chr1B_43208938_G / A,Chr1B_43365653_A / G,Chr1B_43365662_C / G,Chr1B_43365688_C / T,Chr1B_43366627_T / C,Chr1B_43367112_T / G,Chr1B_43367220_C / T,Chr1B_43367247_T / A,Chr1B_43367250_G / A,Chr1B_43367268_G / A,Chr1B_43367343_G / A,Chr1B_43486476_G / A,Chr1B_43486987_G / A,Chr1B_43721968_T / A,Chr1B_43724534_C / T,Chr1B_43726160_A / C,Chr1B_44043034_C / G,Chr1B_44074222_G / C,Chr1B_44074231_T / A,Chr1B_44528425_C / G,Chr1B_44685299_G / C,Chr1B_44685301_A / G,Chr1B_44692217_A / T,Chr1B_44700845_T / A,Chr1B_44700861_T / A,Chr1B_44775277_C / T,Chr1B_45134170_G / C,Chr1B_45134197_C / T,Chr1B_45134246_C / T,Chr1B_45134271_G / A,Chr1B_45207975_T / G,Chr1B_45207977_T / C,Chr1B_45208096_A / C,Chr1B_45648144_C / T,Chr1B_45674023_C / T,Chr1B_45925789_C / T,Chr1B_45925829_T / C,Chr1B_45925862_C / A,Chr1B_45927058_A / G,Chr1B_45927108_G / A,Chr1B_45927134_C / T,Chr1B_46884773_T / C,Chr1B_54544249_C / T,Chr2A_1339133_C / T,Chr2A_17004165_A / G,Chr2A_17331036_C / T,Chr2A_18807941_T / C,Chr2A_18807986_G / A,Chr2A_21855809_A / G,Chr2A_21938533_G / A,Chr2A_21947911_T / C,Chr2A_21956572_G / T,Chr2A_21956591_C / T,Chr2A_21956594_G / A,Chr2A_22073008_C / T,Chr2A_22073019_T / C,Chr2A_22073020_G / A,Chr2A_22084527_T / A,Chr2A_22093230_C / A,Chr2A_22177699_G / T,Chr2A_22432517_T / G,Chr2A_22441656_C / T,Chr2A_22441666_A / G,Chr2A_22441667_G / A,Chr2A_22441704_C / T,Chr2A_22441738_T / G,Chr2A_22441740_C / T,Chr2A_22441789_C / T,Chr2A_22453037_A / C,Chr2A_22453045_G / A,Chr2A_22453110_G / T,Chr2A_22453127_C / T,Chr2A_22453129_C / T,Chr2A_22454646_C / T,Chr2A_22455438_A / T,Chr2A_22455550_G / A,Chr2A_22552814_G / A,Chr2A_22582070_A / T,Chr2A_22587117_G / T,Chr2A_22587138_G / A,Chr2A_22590683_C / T,Chr2A_22590979_A / T,Chr2A_22590992_C / G,Chr2A_22610998_C / T,Chr2A_22623945_G / A,Chr2A_22623953_C / T,Chr2A_22626330_A / G,Chr2A_22629251_C / A,Chr2A_22654482_A / C,Chr2A_22655657_A / T,Chr2A_22655814_C / A,Chr2A_22655821_T / G,Chr2A_22655951_A / T,Chr2A_22656440_T / C,Chr2A_22664493_C / T,Chr2A_22665978_T / C,Chr2A_22666455_T / A,Chr2A_22666472_G / A,Chr2A_22666488_T / C,Chr2A_22666721_T / A,Chr2A_22666999_G / A,Chr2A_22667134_C / T,Chr2A_22694982_G / A,Chr2A_22695005_A / G,Chr2A_22696019_G / A,Chr2A_22696051_C / G,Chr2A_22696052_C / T,Chr2A_22696055_C / T,Chr2A_22696119_G / A,Chr2A_22696140_G / A,Chr2A_22696164_C / T,Chr2A_22712562_G / T,Chr2A_23060073_A / C,Chr2A_23301722_G / A,Chr2A_23316902_G / A,Chr2A_23331772_C / G,Chr2A_23477308_C / T,Chr2A_23478448_C / G,Chr2A_23478468_T / G,Chr2A_23478482_C / A,Chr2A_23479059_A / C,Chr2A_23515383_C / T,Chr2A_23515399_C / A,Chr2A_23515461_G / A,Chr2A_23515476_G / A,Chr2A_23515574_G / A,Chr2A_23515623_T / C,Chr2A_23523624_C / T,Chr2A_23524024_A / T,Chr2A_23527692_G / A,Chr2A_23527744_C / T,Chr2A_23562795_C / T,Chr2A_23562839_A / C,Chr2A_23644776_C / T,Chr2A_23933055_G / T,Chr2A_23933504_T / C,Chr2A_23933577_G / A,Chr2A_23933662_A / C,Chr2A_23993161_A / C,Chr2A_23993416_C / A,Chr2A_24212909_T / C,Chr2A_24212921_A / G,Chr2A_24220945_C / T,Chr2A_24492172_A / G,Chr2A_24493845_A / T,Chr2A_24535170_C / A,Chr2A_24550319_C / T,Chr2A_24550329_G / A,Chr2A_24550426_G / A,Chr2A_24550475_C / T,Chr2A_24550486_G / T,Chr2A_24550641_A / G,Chr2A_24551070_T / A,Chr2A_24570327_T / C,Chr2A_24570354_A / T,Chr2A_24570454_T / C,Chr2A_24570463_C / A,Chr2A_24588798_T / C,Chr2A_24628953_G / C,Chr2A_24628959_A / C,Chr2A_24777641_C / T,Chr2A_24777666_C / T,Chr2A_24777687_C / A,Chr2A_24822275_G / A,Chr2A_24822551_C / A,Chr2A_24829231_C / T,Chr2A_24829236_A / T,Chr2A_24851454_G / A,Chr2A_24858454_A / G,Chr2A_25007959_A / G,Chr2A_25471472_T / G,Chr2A_52830239_A / G,Chr2A_62335175_G / A,Chr2A_62335202_A / G,Chr2A_64361234_A / G,Chr2A_64361252_C / T,Chr2A_64827402_T / C,Chr2A_64872171_C / T,Chr2A_64873313_G / A,Chr2A_64901593_A / T,Chr2A_65295690_G / A,Chr2A_65295735_T / C,Chr2A_65307954_C / T,Chr2A_65307957_T / C,Chr2A_65376433_T / C,Chr2A_65381789_G / A,Chr2A_65381812_A / G,Chr2A_65381816_A / G,Chr2A_65381846_A / G,Chr2A_65381847_C / T,Chr2A_65381881_T / C,Chr2A_65577570_C / T,Chr2A_65577693_C / T,Chr2A_65577730_C / T,Chr2A_65577776_C / A,Chr2A_65577910_A / C,Chr2A_65578070_G / A,Chr2A_65578083_A / G,Chr2A_65578092_T / C,Chr2A_65578093_G / T,Chr2A_65578095_A / G,Chr2A_65585159_A / T,Chr2A_65623669_C / T,Chr2A_65635989_C / T,Chr2A_65692989_G / A,Chr2A_65819814_A / G,Chr2A_65819816_A / G,Chr2A_65843931_A / G,Chr2A_65843933_A / G,Chr2A_65843949_C / T,Chr2A_65844044_A / T,Chr2A_65844052_T / C,Chr2A_65844163_T / G,Chr2A_65844242_T / C,Chr2A_65844588_C / G,Chr2A_65844682_T / C,Chr2A_65844736_T / C,Chr2A_65844743_T / C,Chr2A_65844745_A / G,Chr2A_65844818_T / C,Chr2A_65845203_T / C,Chr2A_65845244_G / A,Chr2A_65845255_A / G,Chr2A_65845287_C / A,Chr2A_65845327_T / C,Chr2A_65845383_G / C,Chr2A_65845392_T / C,Chr2A_65845406_A / G,Chr2A_65845419_T / C,Chr2A_65845429_T / C,Chr2A_65845548_A / G,Chr2A_65845602_A / G,Chr2A_65845614_A / C,Chr2A_65845687_A / G,Chr2A_65845713_T / C,Chr2A_65845725_C / A,Chr2A_65845752_A / C,Chr2A_65845830_A / C,Chr2A_65866330_G / A,Chr2A_65904955_A / T,Chr2A_65908880_T / A,Chr2A_65909057_G / A,Chr2A_65909212_T / C,Chr2A_65909417_A / G,Chr2A_65909469_C / G,Chr2A_65909565_G / T,Chr2A_65909581_T / C,Chr2A_65909607_C / T,Chr2A_65909632_G / A,Chr2A_65909654_A / T,Chr2A_65909758_G / A,Chr2A_65909773_C / T,Chr2A_65909978_T / C,Chr2A_65910010_G / A,Chr2A_65910024_G / A,Chr2A_65910026_A / C,Chr2A_65910050_G / A,Chr2A_65910066_C / T,Chr2A_65910292_G / A,Chr2A_65910479_C / T,Chr2A_65910506_G / A,Chr2A_65910557_G / A,Chr2A_65910608_T / C,Chr2A_65910816_C / T,Chr2A_65963135_C / T,Chr2A_65963285_T / C,Chr2A_65963286_G / A,Chr2A_65974835_T / C,Chr2A_65974880_G / A,Chr2A_65996929_G / A,Chr2A_66006615_T / A,Chr2A_66026413_C / A,Chr2A_66026578_C / G,Chr2A_66122909_A / T,Chr2A_66123702_C / G,Chr2A_66123705_A / G,Chr2A_66123750_C / T,Chr2A_66123763_T / G,Chr2A_66123780_G / T,Chr2A_66123791_G / A,Chr2A_66124689_T / C,Chr2A_66124690_A / C,Chr2A_66124705_G / A,Chr2A_66125591_C / G,Chr2A_66126087_C / T,Chr2A_66126254_T / C,Chr2A_66126288_G / A,Chr2A_66126296_T / C,Chr2A_66126578_A / G,Chr2A_66126752_T / C,Chr2A_66129581_T / A,Chr2A_66129843_C / T,Chr2A_66129856_T / C,Chr2A_66129884_T / A,Chr2A_66129896_G / A,Chr2A_66130710_G / A,Chr2A_66130715_G / A,Chr2A_66133012_T / A,Chr2A_66133039_C / T,Chr2A_66133130_A / G,Chr2A_66133134_C / T,Chr2A_66133452_C / T,Chr2A_66133498_G / C,Chr2A_66133761_A / G,Chr2A_66133794_G / C,Chr2A_66133898_C / T,Chr2A_66138080_C / T,Chr2A_66141241_G / T,Chr2A_66141258_A / G,Chr2A_66141272_T / C,Chr2A_66141886_G / C,Chr2A_66142612_T / C,Chr2A_66142614_G / T,Chr2A_66142620_A / G,Chr2A_66142635_T / G,Chr2A_66196635_C / T,Chr2A_66242970_A / G,Chr2A_66242978_A / T,Chr2A_66243501_G / T,Chr2A_66243504_A / T,Chr2A_66243507_A / T,Chr2A_66243509_A / T,Chr2A_66243514_T / A,Chr2A_66263649_A / G,Chr2A_66263663_A / G,Chr2A_66268212_A / T,Chr2A_66278252_A / G,Chr2A_66278255_G / A,Chr2A_66298536_C / G,Chr2A_66306593_C / T,Chr2A_66306739_C / T,Chr2A_66306756_C / T,Chr2A_66306898_T / C,Chr2A_66311385_G / C,Chr2A_66354597_C / T,Chr2A_66354602_T / C,Chr2A_66359511_A / G,Chr2A_66359646_G / A,Chr2A_66359776_A / T,Chr2A_66362348_A / T,Chr2A_66362473_G / C,Chr2A_66362540_G / A,Chr2A_66362628_C / T,Chr2A_66362756_A / G,Chr2A_66362764_G / A,Chr2A_66362856_A / T,Chr2A_66362860_G / T,Chr2A_66362942_C / A,Chr2A_66362949_G / A,Chr2A_66363127_T / A,Chr2A_66363331_C / T,Chr2A_66368855_A / G,Chr2A_66368902_G / A,Chr2A_66368942_A / T,Chr2A_66371670_G / A,Chr2A_66373395_C / G,Chr2A_66373585_C / G,Chr2A_66373652_A / G,Chr2A_66374053_A / C,Chr2A_66393805_G / A,Chr2A_66401846_G / A,Chr2A_66416677_A / C,Chr2A_66464776_G / A,Chr2A_66495373_C / T,Chr2A_66495377_A / C,Chr2A_66495491_A / G,Chr2A_66495523_C / T,Chr2A_66497552_C / T,Chr2A_66497589_G / A,Chr2A_66497856_T / C,Chr2A_66497892_A / G,Chr2A_66497912_G / T,Chr2A_66497969_A / G,Chr2A_66497978_G / A,Chr2A_66498018_A / G,Chr2A_66498019_C / T,Chr2A_66498058_C / T,Chr2A_66498060_T / C,Chr2A_66498084_T / G,Chr2A_66498087_G / T,Chr2A_66498092_C / T,Chr2A_66498097_G / A,Chr2A_66498150_G / A,Chr2A_66498157_G / A,Chr2A_66498275_G / C,Chr2A_66498335_A / G,Chr2A_66498336_T / C,Chr2A_66501362_C / T,Chr2A_66501420_A / T,Chr2A_66501725_G / T,Chr2A_66501726_C / G,Chr2A_66502551_T / C,Chr2A_66502565_C / T,Chr2A_66502609_A / T,Chr2A_66502623_G / A,Chr2A_66502638_G / A,Chr2A_66502659_G / A,Chr2A_66502696_C / T,Chr2A_66502702_T / A,Chr2A_66502711_C / T,Chr2A_66502716_G / A,Chr2A_66502726_G / A,Chr2A_66502746_A / G,Chr2A_66502760_G / C,Chr2A_66502770_G / A,Chr2A_66502798_G / A,Chr2A_66502907_G / A,Chr2A_66502924_C / T,Chr2A_66503012_A / T,Chr2A_66503067_A / C,Chr2A_66503115_C / T,Chr2A_66503183_C / T,Chr2A_66503186_T / G,Chr2A_66503190_C / T,Chr2A_66503202_C / G,Chr2A_66503242_C / T,Chr2A_66503305_G / A,Chr2A_66503313_T / A,Chr2A_66503328_C / T,Chr2A_66503717_G / A,Chr2A_66503812_C / T,Chr2A_66503823_T / G,Chr2A_66504233_T / G,Chr2A_66504263_C / A,Chr2A_66504264_C / A,Chr2A_66504284_G / C,Chr2A_66506552_T / C,Chr2A_66506616_G / T,Chr2A_66521691_G / A,Chr2A_66521695_A / G,Chr2A_66521980_A / T,Chr2A_66558963_A / G,Chr2A_66560711_T / A,Chr2A_66560715_C / T,Chr2A_66560734_A / T,Chr2A_66582309_C / G,Chr2A_66582513_T / C,Chr2A_66582517_C / A,Chr2A_66583153_G / T,Chr2A_66583836_A / T,Chr2A_66602095_A / C,Chr2A_66622961_G / A,Chr2A_66632566_A / T,Chr2A_66649978_C / G,Chr2A_66688977_A / G,Chr2A_66688993_G / C,Chr2A_66689020_T / G,Chr2A_66699411_C / T,Chr2A_66699851_A / G,Chr2A_66701157_C / T,Chr2A_66701174_T / C,Chr2A_66701229_T / C,Chr2A_66701961_G / A,Chr2A_66702950_C / T,Chr2A_66703015_C / T,Chr2A_66703024_G / A,Chr2A_66729693_C / A,Chr2A_66770831_T / A,Chr2A_66774364_A / G,Chr2A_66774377_C / T,Chr2A_66785604_C / T,Chr2A_66789031_C / T,Chr2A_66789048_G / A,Chr2A_66789350_C / T,Chr2A_66789363_A / T,Chr2A_66789521_T / G,Chr2A_66789530_C / G,Chr2A_66789562_A / T,Chr2A_66810490_T / G,Chr2A_66810510_T / C,Chr2A_66810535_A / T,Chr2A_66817549_T / A,Chr2A_66817590_T / C,Chr2A_66825137_G / T,Chr2A_66833318_C / T,Chr2A_66884959_T / C,Chr2A_66884961_C / T,Chr2A_66926783_C / T,Chr2A_66930195_A / G,Chr2A_66936223_G / A,Chr2A_66937543_A / T,Chr2A_66937568_A / G,Chr2A_66939070_C / A,Chr2A_66939102_G / A,Chr2A_66952798_A / G,Chr2A_66952806_C / T,Chr2A_66952821_G / T,Chr2A_66952826_G / A,Chr2A_66960201_C / G,Chr2A_66964733_T / C,Chr2A_66965218_C / A,Chr2A_66965303_C / T,Chr2A_66965322_T / C,Chr2A_66965330_A / G,Chr2A_66967124_G / T,Chr2A_66967127_G / A,Chr2A_66973746_C / T,Chr2A_66974406_A / G,Chr2A_66974437_C / A,Chr2A_66974441_C / T,Chr2A_66974710_A / C,Chr2A_66974713_A / C,Chr2A_66974718_G / A,Chr2A_66974732_C / T,Chr2A_66974844_T / A,Chr2A_66974891_G / A,Chr2A_66974896_T / C,Chr2A_66974911_T / C,Chr2A_66974986_G / C,Chr2A_66975010_T / G,Chr2A_66975014_A / T,Chr2A_66975033_T / C,Chr2A_66975095_T / C,Chr2A_66975118_A / C,Chr2A_66975166_G / A,Chr2A_66987096_G / A,Chr2A_66987106_G / T,Chr2A_66987121_G / C,Chr2A_66987576_G / T,Chr2A_66987591_G / T,Chr2A_66987992_C / G,Chr2A_66988016_G / T,Chr2A_66988032_G / A,Chr2A_66988057_G / A,Chr2A_66988127_A / G,Chr2A_66988135_G / A,Chr2A_66989214_G / A,Chr2A_67033879_C / A,Chr2A_67033892_G / T,Chr2A_67053281_T / G,Chr2A_67101163_G / A,Chr2A_67101357_G / A,Chr2A_67101429_C / T,Chr2A_67147341_G / T,Chr2A_67246253_T / C,Chr2A_67261854_T / C,Chr2A_67261900_C / A,Chr2A_67273093_T / C,Chr2A_67273121_A / G,Chr2A_67323380_C / T,Chr2A_67324506_T / G,Chr2A_67324523_A / T,Chr2A_67342360_G / T,Chr2A_67357986_A / T,Chr2A_67357987_C / T,Chr2A_67357988_A / T,Chr2A_67360984_C / T,Chr2A_67437965_A / G,Chr2A_67437998_G / A,Chr2A_67441982_C / G,Chr2A_67442022_G / A,Chr2A_67442127_C / T,Chr2A_67442131_T / C,Chr2A_67442149_T / C,Chr2A_67442155_C / T,Chr2A_67449306_G / A,Chr2A_67449308_G / A,Chr2A_67449310_A / G,Chr2A_67449312_C / T,Chr2A_67449318_A / G,Chr2A_67449319_C / T,Chr2A_67449346_G / A,Chr2A_67484933_G / A,Chr2A_67484957_A / C,Chr2A_67499327_A / G,Chr2A_67499361_A / G,Chr2A_67499468_T / C,Chr2A_67499549_C / T,Chr2A_67499574_C / T,Chr2A_67499593_A / T,Chr2A_67499644_C / T,Chr2A_67499758_C / A,Chr2A_67499770_T / C,Chr2A_67499778_C / T,Chr2A_67499794_T / C,Chr2A_67499856_G / A,Chr2A_67500321_A / G,Chr2A_67500323_C / T,Chr2A_67505622_G / A,Chr2A_67689893_G / A,Chr2A_67689932_G / A,Chr2A_67689933_T / A,Chr2A_67690031_A / G,Chr2A_67690212_T / C,Chr2A_67690612_C / A,Chr2A_67690638_T / C,Chr2A_67690646_A / T,Chr2A_67690881_C / G,Chr2A_67690901_T / G,Chr2A_67690906_G / T,Chr2A_67700440_A / G,Chr2A_67700451_G / A,Chr2A_67700548_C / G,Chr2A_67700700_G / A,Chr2A_67700702_A / G,Chr2A_67700730_T / A,Chr2A_67700735_C / A,Chr2A_67700767_C / T,Chr2A_67700857_C / G,Chr2A_67700916_A / C,Chr2A_67700924_G / C,Chr2A_67700955_T / C,Chr2A_67700968_G / A,Chr2A_67700994_A / G,Chr2A_67701045_T / A,Chr2A_67701061_C / A,Chr2A_67701106_G / A,Chr2A_67701108_C / A,Chr2A_67701331_A / G,Chr2A_67701377_G / A,Chr2A_67701380_A / G,Chr2A_67701962_G / A,Chr2A_67702115_T / A,Chr2A_67702121_G / A,Chr2A_67702148_A / G,Chr2A_67702157_G / A,Chr2A_67702698_T / C,Chr2A_67702950_C / A,Chr2A_67703004_T / C,Chr2A_67703023_C / G,Chr2A_67703195_T / C,Chr2A_67703438_C / A,Chr2A_67703511_T / G,Chr2A_67703542_T / A,Chr2A_67707255_T / C,Chr2A_67710270_C / A,Chr2A_67710281_A / G,Chr2A_67710285_A / G,Chr2A_67723848_T / A,Chr2A_67922243_G / A,Chr2A_67936108_C / T,Chr2A_67945744_G / A,Chr2A_67947210_T / C,Chr2A_67948880_T / C,Chr2A_67948890_G / A,Chr2A_67948938_T / C,Chr2A_67949103_A / C,Chr2A_67949115_C / T,Chr2A_67949119_T / C,Chr2A_67960580_T / G,Chr2A_67960729_G / A,Chr2A_67960736_G / C,Chr2A_67960821_G / A,Chr2A_67960842_C / T,Chr2A_67960867_G / A,Chr2A_67960884_C / T,Chr2A_67961046_C / A,Chr2A_67961087_A / G,Chr2A_67961127_C / T,Chr2A_67961168_A / G,Chr2A_67961178_T / G,Chr2A_67961186_G / A,Chr2A_67961250_T / C,Chr2A_67961259_T / G,Chr2A_67961286_T / G,Chr2A_67964127_T / C,Chr2A_67964130_C / T,Chr2A_67964139_C / T,Chr2A_67964146_T / C,Chr2A_67964152_A / G,Chr2A_67964160_G / A,Chr2A_67964169_G / A,Chr2A_67964193_A / T,Chr2A_68205323_C / T,Chr2A_68207319_G / T,Chr2A_69490501_A / C,Chr2A_69490642_T / C,Chr2A_69490643_G / A,Chr2A_77462850_C / A,Chr2A_79148333_C / A,Chr2A_79410445_A / G,Chr2A_79410495_A / T,Chr2A_79410499_A / G,Chr2A_79410506_G / A,Chr2A_79479988_A / G,Chr2A_79480042_C / T,Chr2A_79480059_A / G,Chr2A_79488008_A / G,Chr2A_83203595_T / C,Chr2A_83290598_G / T,Chr2A_83313086_A / G,Chr2A_83369579_A / T,Chr2A_83369643_A / C,Chr2A_83385112_A / T,Chr2A_83385158_G / A,Chr2A_83385907_C / T,Chr2A_83387330_C / G,Chr2A_83736086_A / C,Chr2A_83757921_C / A,Chr2A_83757995_T / C,Chr2A_83980746_A / G,Chr2A_83980751_G / C,Chr2A_83980843_T / C,Chr2A_83989866_G / T,Chr2A_83989946_T / A,Chr2A_83989947_C / A,Chr2A_83990186_C / T,Chr2A_83990383_T / C,Chr2A_83990417_C / T,Chr2A_83990499_G / A,Chr2A_83990597_C / T,Chr2A_83991729_A / C,Chr2A_83991773_C / A,Chr2A_83992201_G / A,Chr2A_83994252_G / A,Chr2A_84191328_G / A,Chr2A_84191583_G / A,Chr2A_84245898_T / C,Chr2A_84254780_G / A,Chr2A_84254783_G / T,Chr2A_84305167_T / C,Chr2A_84335744_G / A,Chr2A_84386573_T / C,Chr2A_84386597_C / T,Chr2A_84539162_A / G,Chr2A_84698188_A / C,Chr2A_84825533_G / A,Chr2A_84865221_A / T,Chr2A_85043605_A / T,Chr2A_85131482_A / G,Chr2A_85243407_A / G,Chr2A_85403859_G / A,Chr2A_85471810_T / A,Chr2A_85553081_A / G,Chr2A_85553127_C / T,Chr2A_85553144_T / G,Chr2A_85553151_A / G,Chr2A_85660308_C / T,Chr2A_85660311_T / A,Chr2A_85660325_T / C,Chr2A_85691341_C / A,Chr2A_85694911_A / T,Chr2A_85699914_T / C,Chr2A_85699923_A / G,Chr2A_85699924_T / C,Chr2A_85700106_C / T,Chr2A_85700288_A / G,Chr2A_85701866_A / G,Chr2A_85705706_C / T,Chr2A_85718365_C / A,Chr2A_85718407_T / A,Chr2A_85740874_G / T,Chr2A_85744889_C / T,Chr2A_85744928_A / T,Chr2A_85770759_T / G,Chr2A_85775450_T / G,Chr2A_85775466_G / C,Chr2A_85800086_G / A,Chr2A_85800869_T / C,Chr2A_85800907_A / T,Chr2A_85806406_G / A,Chr2A_85806412_C / T,Chr2A_85806420_A / G,Chr2A_85806491_C / G,Chr2A_85806550_C / A,Chr2A_85806748_A / G,Chr2A_85806753_T / C,Chr2A_85806818_C / T,Chr2A_85807058_T / A,Chr2A_85829383_C / T,Chr2A_85840528_C / T,Chr2A_85848277_A / G,Chr2A_85863418_G / A,Chr2A_85863473_A / C,Chr2A_85867415_T / C,Chr2A_85949066_C / A,Chr2A_85949131_A / C,Chr2A_86017599_C / A,Chr2A_86027521_G / A,Chr2A_86027522_A / T,Chr2A_86028594_A / G,Chr2A_86046909_T / G,Chr2A_86127102_T / A,Chr2A_86127382_T / G,Chr2A_86127390_C / T,Chr2A_86131831_T / A,Chr2A_86135853_T / C,Chr2A_86135942_T / A,Chr2A_86136158_T / C,Chr2A_86137410_C / T,Chr2A_86147446_G / A,Chr2A_86147512_C / T,Chr2A_86156419_A / G,Chr2A_86165443_A / T,Chr2A_86208695_C / T,Chr2A_86208696_C / A,Chr2A_86208707_T / C,Chr2A_86217601_T / C,Chr2A_86217664_A / T,Chr2A_86254514_A / T,Chr2A_86255255_C / T,Chr2A_86255465_G / A,Chr2A_86256150_A / G,Chr2A_86256216_C / T,Chr2A_86258949_G / A,Chr2A_86259040_C / A,Chr2A_86283248_A / T,Chr2A_86492440_A / G,Chr2A_86492450_G / C,Chr2A_86493641_G / A,Chr2B_7595902_C / T,Chr2B_9243020_C / T,Chr2B_18136856_G / A,Chr2B_18136867_T / G,Chr2B_20286964_T / A,Chr2B_22286959_C / G,Chr2B_32531061_C / T,Chr2B_33176780_A / G,Chr2B_44539083_G / T,Chr2B_54579465_C / T,Chr2B_54579615_C / T,Chr2B_62283365_G / C,Chr2B_62283417_G / C,Chr2B_62286782_A / G,Chr2B_62286883_G / A,Chr2B_62293045_A / T,Chr2B_62293106_T / A,Chr2B_62293155_A / G,Chr2B_62293393_G / T,Chr2B_62293413_C / T,Chr2B_62293422_A / C,Chr2B_62293519_G / A,Chr2B_62293551_C / G,Chr2B_62293573_C / T,Chr2B_62293615_A / G,Chr2B_62293625_A / T,Chr2B_62293636_A / T,Chr2B_62293960_G / A,Chr2B_62326898_T / A,Chr2B_62331447_C / A,Chr2B_62331450_T / G,Chr2B_62362932_C / T,Chr2B_62363194_A / G,Chr2B_62363225_T / C,Chr2B_62363244_A / G,Chr2B_62363294_A / G,Chr2B_62363516_C / G,Chr2B_62581017_A / C,Chr2B_62946525_A / G,Chr2B_62946538_A / G,Chr2B_62946680_T / C,Chr2B_62950390_C / T,Chr2B_63021796_A / T,Chr2B_63021846_A / G,Chr2B_63021857_A / G,Chr2B_63107436_T / C,Chr2B_63108604_C / T,Chr2B_63145010_A / G,Chr2B_63442552_T / A,Chr2B_63631946_A / G,Chr2B_63631949_T / C,Chr2B_63631956_T / A,Chr2B_63716424_C / T,Chr2B_63716461_G / A,Chr2B_63792073_G / T,Chr2B_63863924_C / G,Chr2B_63863937_G / A,Chr2B_63892181_G / T,Chr2B_63924256_T / A,Chr2B_63934513_A / C,Chr2B_63934637_A / C,Chr2B_63954064_C / T,Chr2B_63954106_C / A,Chr2B_63954110_A / G,Chr2B_63995236_A / T,Chr2B_63995329_T / A,Chr2B_63995359_T / A,Chr2B_63996613_A / G,Chr2B_63996615_A / G,Chr2B_63998775_T / C,Chr2B_63998922_A / T,Chr2B_63998955_G / A,Chr2B_63998997_C / A,Chr2B_63999000_T / G,Chr2B_64023718_C / G,Chr2B_64023779_T / C,Chr2B_64023788_G / T,Chr2B_64026019_A / C,Chr2B_64030459_C / T,Chr2B_64149988_T / C,Chr2B_64150009_C / A,Chr2B_64150056_C / G,Chr2B_64150074_C / G,Chr2B_64150125_T / C,Chr2B_64205480_A / T,Chr2B_64337440_T / A,Chr2B_64375514_A / T,Chr2B_64375830_C / A,Chr2B_64375928_G / A,Chr2B_64376043_C / T,Chr2B_64376058_C / T,Chr2B_64376479_G / A,Chr2B_64376680_C / T,Chr2B_64376687_G / A,Chr2B_64376820_C / T,Chr2B_64379986_G / A,Chr2B_64380016_A / G,Chr2B_64380205_C / T,Chr2B_64380241_G / A,Chr2B_64380269_G / A,Chr2B_64380326_A / G,Chr2B_64380365_G / A,Chr2B_64380400_G / T,Chr2B_64380402_C / T,Chr2B_64380430_G / A,Chr2B_64380552_G / T,Chr2B_64380653_G / A,Chr2B_64380675_G / A,Chr2B_64380707_G / A,Chr2B_64380758_C / A,Chr2B_64381082_G / A,Chr2B_64381103_G / T,Chr2B_64381313_C / T,Chr2B_64381316_C / T,Chr2B_64381340_C / A,Chr2B_64526752_C / T,Chr2B_64526761_C / A,Chr2B_64527445_T / C,Chr2B_64527451_C / T,Chr2B_64638498_A / G,Chr2B_64638501_A / C,Chr2B_64644739_A / G,Chr2B_64647653_G / A,Chr2B_64649464_C / T,Chr2B_64649482_T / G,Chr2B_64649608_G / A,Chr2B_65197985_G / A,Chr2B_65469937_T / C,Chr2B_69134895_G / A,Chr2B_75122406_A / C,Chr2B_75344354_C / T,Chr2B_77402954_G / C,Chr2B_80328564_C / T,Chr2B_80828780_G / A,Chr2B_80936137_T / C,Chr2B_81232003_G / A,Chr2B_81232126_G / A,Chr2B_81239815_T / G,Chr2B_81334785_G / T,Chr2B_81338734_C / T,Chr2B_81345224_A / T,Chr2B_81409154_T / A,Chr2B_81419561_C / A,Chr2B_81421826_C / A,Chr2B_81422412_T / G,Chr2B_81424855_A / C,Chr2B_81424934_G / A,Chr2B_81425784_A / C,Chr2B_81425802_C / A,Chr2B_81425980_C / T,Chr2B_81426028_T / C,Chr2B_82015647_C / T,Chr2B_82057366_G / T,Chr2B_82081664_T / C,Chr2B_82292899_G / T,Chr2B_82366417_A / G,Chr2B_82785817_T / C,Chr2B_82787315_T / G,Chr2B_83066965_C / T,Chr2B_83077507_A / T,Chr2B_83572866_A / G,Chr2B_84131203_G / C,Chr3A_21707094_C / G,Chr3A_21707097_C / G,Chr3A_21707102_A / G,Chr3A_21707103_C / A,Chr3A_21707130_A / T,Chr3A_21707136_A / T,Chr3A_21707140_C / A,Chr3A_21707146_C / G,Chr3A_21707149_T / G,Chr3A_21707154_A / G,Chr3A_21707266_T / A,Chr3A_21707271_A / G,Chr3A_31296192_C / A,Chr3A_31499680_A / G,Chr4A_14607459_C / G,Chr4A_14607467_T / C,Chr4A_14607482_A / C,Chr4A_14607486_A / T,Chr4A_14607505_T / A,Chr4A_33515569_A / G,Chr4A_34421064_C / G,Chr4A_35186034_G / A,Chr4A_49671519_T / A,Chr4A_49671522_T / C,Chr4A_49671532_G / A,Chr4A_66109263_T / G,Chr4B_3113532_T / G,Chr4B_4419403_C / T,Chr4B_4864511_T / C,Chr4B_5266843_A / T,Chr4B_55445501_C / T,Chr5A_15051750_C / T,Chr5A_19254188_A / T,Chr5A_20192685_G / A,Chr5A_20258846_T / C,Chr5A_20259257_C / T,Chr5A_20269559_C / T,Chr5A_20269584_G / A,Chr5A_20269611_T / C,Chr5A_20281598_T / A,Chr5A_20281641_G / A,Chr5A_20299478_A / T,Chr5A_20299572_T / G,Chr5A_20299670_C / A,Chr5A_20299826_G / A,Chr5A_20300898_C / T,Chr5A_20406145_C / T,Chr5A_20536458_C / G,Chr5A_20536465_A / G,Chr5A_20536473_T / A,Chr5A_20536476_G / A,Chr5A_20536613_A / G,Chr5A_20536621_A / G,Chr5A_20537276_G / C,Chr5A_20537415_C / T,Chr5A_20596792_A / G,Chr5A_20972159_G / A,Chr5A_20973194_C / T,Chr5A_20973202_C / T,Chr5A_20974614_G / T,Chr5A_20974676_A / C,Chr5A_20974688_C / G,Chr5A_20974920_C / G,Chr5A_20974928_G / A,Chr5A_20974965_G / T,Chr5A_20975001_T / C,Chr5A_20975003_T / A,Chr5A_20975005_T / A,Chr5A_20976997_C / T,Chr5A_20977885_C / T,Chr5A_20978375_C / A,Chr5A_20978471_C / T,Chr5A_20978605_A / G,Chr5A_20978613_G / A,Chr5A_20990425_A / G,Chr5A_20990482_A / G,Chr5A_20990524_A / G,Chr5A_20990532_T / C,Chr5A_20990533_G / A,Chr5A_20990534_G / A,Chr5A_20990549_G / T,Chr5A_20990550_C / G,Chr5A_21010593_G / T,Chr5A_21094373_C / T,Chr5A_21094508_T / C,Chr5A_21094533_G / A,Chr5A_21094536_G / A,Chr5A_21108096_T / C,Chr5A_21108854_T / A,Chr5A_21117534_C / T,Chr5A_21117544_C / T,Chr5A_21203896_C / T,Chr5A_21489052_G / T,Chr5A_21580701_G / A,Chr5A_21596255_G / T,Chr5A_21596268_C / T,Chr5A_23435054_G / A,Chr5A_31014330_A / G,Chr5A_33357516_C / A,Chr5A_74251316_A / C,Chr5A_76214067_A / G,Chr5A_76245190_T / G,Chr5A_76245374_C / T,Chr5A_76245387_C / T,Chr5A_76245685_T / C,Chr5A_76745283_C / T,Chr5A_77133652_C / T,Chr5A_77146950_C / A,Chr5A_77147047_C / G,Chr5A_77148493_G / A,Chr5A_77186753_C / T,Chr5A_77186833_T / C,Chr5A_77186838_G / A,Chr5A_77186844_T / G,Chr5A_77361976_G / C,Chr5A_77655957_T / C,Chr5A_78394334_T / C,Chr5A_78394366_T / C,Chr5A_78394434_C / T,Chr5A_78394436_C / T,Chr5A_78636949_G / T,Chr5A_78713534_T / A,Chr5A_78815771_T / C,Chr5A_78961378_C / G,Chr5A_78961922_C / A,Chr5A_78961974_G / A,Chr5A_78961995_A / C,Chr5A_78962013_G / A,Chr5A_78962330_G / A,Chr5A_78971043_T / C,Chr5A_78971198_T / C,Chr5A_78971603_C / G,Chr5A_78971698_C / T,Chr5A_78971782_A / G,Chr5A_78972176_T / G,Chr5A_78972191_T / C,Chr5A_79044862_A / G,Chr5A_79044866_A / T,Chr5A_79045234_T / A,Chr5A_79045332_T / C,Chr5A_79045361_C / T,Chr5A_79045375_G / A,Chr5A_79123820_C / T,Chr5A_79248545_A / T,Chr5A_79518435_C / T,Chr5A_79528046_G / A,Chr5A_79535916_T / C,Chr5A_79571650_A / G,Chr5A_79982959_G / C,Chr5A_80126010_C / A,Chr5A_80435914_T / A,Chr5A_80681797_A / G,Chr5A_80681812_C / A,Chr5A_80681973_A / G,Chr5A_80684818_T / C,Chr5A_80684918_A / T,Chr5A_80745900_C / T,Chr5A_80756378_T / C,Chr5A_80797881_T / C,Chr5A_80808060_T / C,Chr5A_80857985_C / T,Chr5A_80857989_C / T,Chr5A_81217913_A / C,Chr5A_81217981_C / G,Chr5A_81217993_G / A,Chr5A_81218028_G / T,Chr5A_81218170_T / G,Chr5A_81218251_C / T,Chr5A_81354626_C / G,Chr5A_81473741_A / T,Chr5A_81509566_C / T,Chr5A_81509823_G / A,Chr5A_81509921_G / A,Chr5A_81548430_G / A,Chr5A_81556151_G / A,Chr5A_81744071_G / A,Chr5A_81747479_C / A,Chr5A_81747810_A / T,Chr5A_81747826_A / G,Chr5A_81747954_A / T,Chr5A_81747956_A / T,Chr5A_81747970_G / T,Chr5A_81751391_G / T,Chr5A_81751397_C / T,Chr5A_81755082_G / A,Chr5A_81759038_T / C,Chr5A_81769987_G / A,Chr5A_82560459_C / A,Chr5A_85058524_T / C,Chr5A_85077996_G / C,Chr5A_85129188_C / T,Chr5A_85598728_C / T,Chr5A_857 94633_C / T,Chr5A_85952857_A / C,Chr5A_89540375_T / C,Chr5A_89540395_T / C,Chr5A_89540400_T / C,Chr5A_89540405_C / T,Chr5A_89589057_A / T, Chr5B_17867240_T / C,Chr5B_17867243_C / T,Chr5B_17867529_A / G,Chr5B_17867538_A / G,Chr5B_17867561_A / G,Chr5B_17867667_A / T,Chr5B_18018518_C / G,Chr5B_18018816_A / G,Chr5B_18020799_C / T,Chr5B_18045745_A / G,Chr5B_18645807_C / T,Chr5B_18826608_C / T,Chr5B_19282333_C / A, Chr5B_19695844_T / C,Chr5B_19695863_C / T,Chr5B_19766656_G / A,Chr5B_19766662_G / A,Chr5B_19816401_A / G,Chr5B_19817064_T / G,Chr5B_19861189_C / T,Chr5B_20116060_C / T,Chr5B_20116203_G / A,Chr5B_20144084_C / T,Chr5B_20153910_A / T,Chr5B_20153921_T / C,Chr5B_20154026_A / G, Chr5B_20154290_C / T,Chr5B_20206639_G / A,Chr5B_30339803_G / A,Chr5B_30339816_G / A,Chr5B_30584791_C / T,Chr5B_31928686_C / G,Chr5B_319 28694_A / G,Chr5B_53855683_C / T,Chr5B_53855703_T / C,Chr5B_53941504_G / A,Chr5B_53941671_T / G,Chr5B_54900291_G / A,Chr5B_54900322_G / A,Chr5B_55108475_C / G,Chr5B_55109194_T / G,Chr5B_55109306_C / T,Chr5B_55247890_A / G,Chr5B_66916889_T / G,Chr5B_69002627_T / C,Chr5B_74045937_C / T,Chr5B_74048129_T / A,Chr5B_74048132_G / A,Chr5B_74306748_C / T,Chr5B_74321711_T / A,Chr5B_74472049_C / T,Chr5B_74472090_C / T,Chr5B_74605105_T / A,Chr5B_74605126_A / G,Chr5B_74606787_A / G,Chr5B_74607238_G / T,Chr5B_77228700_T / C,Chr5B_77415560_G / A,Chr5B_77680797_T / C,Chr5B_82461255_C / T,Chr5B_84451021_C / T,Chr5B_85330711_C / A,Chr6A_5693656_C / T,Chr6A_7509618_G / A,Chr6A_7516325_C / T,Chr6A_7520207_C / A,Chr6A_7571723_C / A,Chr6A_7824382_C / T,Chr6A_7874843_G / A,Chr6A_7906316_A / C,Chr6A_7994431_T / A,Chr6A_10514367_G / A,Chr6A_23803122_T / C,Chr6A_28190185_A / G,Chr6A_32719714_A / G,Chr6A_33374167_A / G,Chr6A_33803758_A / G,Chr6A_33848006_C / A,Chr6A_34084402_A / C,Chr6A_34472908_A / C,Chr6A_34472996_G / A,Chr6A_34615635_C / G,Chr6A_35105127_G / A,Chr6A_35105130_A / G,Chr6A_35105205_T / C,Chr6A_35105259_A / C,Chr6A_35723439_T / A,Chr6A_35723459_T / G,Chr6A_35725765_T / C,Chr6A_35727054_T / C,Chr6A_35727102_T / C,Chr6A_36986743_T / C,Chr6A_48292184_T / A,Chr6A_56895284_C / T,Chr6A_57749037_G / C,Chr6A_57749067_G / C,Chr6A_57749074_T / C,Chr6A_57749092_C / A,Chr6A_57749097_G / T,Chr6A_62603318_C / T,Chr6A_62746712_G / A,Chr6A_62755651_T / C,Chr6A_62837380_G / A,Chr6A_62892985_T / C,Chr6A_62952420_C / T,Chr6A_63082146_C / T,Chr6A_63494745_G / A,Chr6A_63509435_T / C,Chr6A_63539500_T / C,Chr6A_63556309_T / C,Chr6A_63556454_T / C,Chr6A_63556703_C / T,Chr6A_63558115_G / A,Chr6A_63558143_C / T,Chr6A_63559908_A / G,Chr6A_63559925_C / T,Chr6A_63568626_G / T,Chr6A_63619535_C / A,Chr6A_63694789_A / G,Chr6A_63708116_C / T,Chr6A_63708122_A / G,Chr6A_63708247_C / T,Chr6A_63708896_C / T,Chr6A_63708925_T / A,Chr6A_63709873_G / A,Chr6A_63711550_T / C,Chr6A_63711552_G / A,Chr6A_63711572_G / A,Chr6A_63711577_A / G,Chr6A_63729769_T / C,Chr6A_63749702_C / A,Chr6A_63955194_G / A,Chr6A_63968507_T / C,Chr6A_64014158_A / C,Chr6A_64014377_C / T,Chr6A_64014384_A / T,Chr6A_64018144_T / C,Chr6A_64065654_T / G,Chr6A_64089052_C / A,Chr6A_64089065_A / T,Chr6A_64089081_G / C,Chr6A_64129753_T / G,Chr6A_64129778_G / A,Chr6A_64129788_C / T,Chr6A_64146599_C / T,Chr6A_64147204_T / G,Chr6A_64147222_G / A,Chr6A_64147432_G / A,Chr6A_64147454_C / T,Chr6A_64147496_T / C,Chr6A_64147568_G / A,Chr6A_64147579_T / G,Chr6A_64147587_C / T,Chr6A_64148745_G / T,Chr6A_64160084_A / T,Chr6A_64160149_C / T,Chr6A_64161185_T / A,Chr6A_64162430_A / G,Chr6A_64162511_G / T,Chr6A_64179945_A / G,Chr6A_64180112_T / C,Chr6A_64180117_C / T,Chr6A_64201129_C / T,Chr6A_64201358_G / A,Chr6A_64201367_C / T,Chr6A_64201381_C / T,Chr6A_64202146_G / A,Chr6A_64209425_G / A,Chr6A_64209471_T / C,Chr6A_64267134_A / T,Chr6A_64290145_G / A,Chr6A_64290217_C / T,Chr6A_64290248_C / T,Chr6A_64290579_C / T,Chr6A_64312740_G / A,Chr6A_64315382_A / C,Chr6A_64315386_T / C,Chr6A_64315496_C / T,Chr6A_64315628_C / G,Chr6A_64315775_G / A,Chr6A_64315782_T / C,Chr6A_64315844_C / T,Chr6A_64316410_G / A,Chr6A_64323599_T / C,Chr6A_64325861_A / G,Chr6A_64325943_G / A,Chr6A_64362054_C / T,Chr6A_64364558_C / T,Chr6A_64462109_G / A,Chr6A_64474018_A / G,Chr6A_65189489_T / A,Chr6A_65282097_G / A,Chr6A_65460066_G / C,Chr6A_66169829_C / T,Chr6A_66217634_G / A,Chr6A_66490103_T / G,Chr6A_66490142_G / A,Chr6A_67147650_C / T,Chr6A_67220087_T / G,Chr6A_67311017_C / T,Chr6A_68456880_A / G,Chr6B_4325212_G / T,Chr6B_4847775_C / A,Chr6B_6609426_G / T,Chr6B_6771187_A / T,Chr6B_11487741_T / C,Chr6B_11975072_C / G,Chr6B_12318905_C / T,Chr6B_12946938_C / T,Chr6B_12949566_C / T,Chr6B_12949597_T / C,Chr6B_13883766_A / T,Chr6B_14242589_A / C,Chr6B_14528883_A / G,Chr6B_14770825_A / G,Chr6B_18361661_C / A,Chr6B_18911444_C / A,Chr6B_19526750_G / C,Chr6B_19911569_A / G,Chr6B_20266043_G / T,Chr6B_23739373_C / A,Chr6B_25662011_A / C,Chr6B_25985348_G / T,Chr6B_25989154_A / G,Chr6B_26050033_T / C,Chr6B_26543473_C / T,Chr6B_26570395_A / T,Chr6B_26571017_A / C,Chr6B_31663576_A / G,Chr6B_31721317_C / T,Chr6B_32169890_A / G,Chr6B_32169910_G / T,Chr6B_32171826_C / A,Chr6B_32173240_T / C,Chr6B_32214694_G / A,Chr6B_32214858_C / T,Chr6B_32232106_T / C,Chr6B_32232799_C / T,Chr6B_32233972_A / C,Chr6B_32254175_C / G,Chr6B_32442378_A / G,Chr6B_32442388_C / T,Chr6B_32442398_A / G,Chr6B_32564949_A / G,Chr6B_32609563_C / T,Chr6B_32843726_C / G,Chr6B_33137971_C / G,Chr6B_33137973_C / T,Chr6B_33196441_A / G,Chr6B_33196614_T / C,Chr6B_33196674_T / C,Chr6B_33214263_T / C,Chr6B_33214319_G / T,Chr6B_33384504_A / C,Chr6B_33523882_A / G,Chr6B_33524341_G / A,Chr6B_33573770_G / A,Chr6B_33573780_C / T,Chr6B_33573800_T / A,Chr6B_33573806_T / C,Chr6B_33573853_T / C,Chr6B_33573912_T / C,Chr6B_33573978_A / G,Chr6B_33573991_T / C,Chr6B_33573995_A / G,Chr6B_33574285_T / C,Chr6B_33575584_A / G,Chr6B_33575647_G / A,Chr6B_33575746_T / C,Chr6B_33576775_A / G,Chr6B_33576791_T / A,Chr6B_34018441_G / A,Chr6B_34018945_T / G,Chr6B_34100199_T / G,Chr6B_34359886_A / T,Chr6B_34398836_T / G,Chr6B_34403318_A / T,Chr6B_34409269_A / C,Chr6B_34409296_C / A,Chr6B_34409299_T / G,Chr6B_34448255_G / A,Chr6B_34487985_G / A,Chr6B_34487995_T / C,Chr6B_34487996_T / G,Chr6B_34488004_T / G,Chr6B_34488005_G / C,Chr6B_34488179_A / G,Chr6B_34557817_C / A,Chr6B_34666117_C / T,Chr6B_34666690_T / C,Chr6B_35008381_T / C,Chr6B_35010204_T / A,Chr6B_35010206_A / G,Chr6B_35010207_A / G,Chr6B_35115243_T / C,Chr6B_35123606_T / C,Chr6B_35237047_T / C,Chr6B_35306212_C / G,Chr6B_49616231_A / G,Chr6B_56736395_C / T,Chr6B_58825298_A / G,Chr6B_58977821_C / T,Chr6B_58977822_C / T,Chr6B_59590903_C / T,Chr6B_59952552_G / A,Chr6B_60118099_C / G,Chr6B_60118101_T / A,Chr6B_60118112_T / C,Chr6B_60124531_A / G,Chr6B_60169214_T / G,Chr6B_60384887_G / C,Chr6B_61487225_T / C,Chr7A_4010260_G / A,Chr7A_6787129_C / T,Chr7A_6787131_A / G,Chr7A_6877736_G / C,Chr7A_6932258_G / A,Chr7A_6932262_C / T,Chr7A_7099517_G / A,Chr7A_7099574_G / A,Chr7A_7347235_C / T,Chr7A_7347280_C / T,Chr7A_7347281_T / C,Chr7A_11805037_A / T,Chr7A_11806349_G / A,Chr7A_20401198_T / C,Chr7A_20401216_A / T,Chr7A_24669884_C / T,Chr7A_24835244_C / T,Chr7A_24842488_T / G,Chr7A_24846097_T / G,Chr7A_29939357_A / G,Chr7A_30352532_A / C,Chr7A_30741443_A / G,Chr7A_30741452_A / T,Chr7A_30741489_T / C,Chr7A_30741506_T / G,Chr7A_30741952_G / A,Chr7A_30742119_C / T,Chr7A_30804617_G / C,Chr7A_30880032_G / C,Chr7A_30881362_C / T,Chr7A_30888667_G / A,Chr7A_30888672_G / A,Chr7A_30891398_C / A,Chr7A_30891401_T / C,Chr7A_30891523_C / T,Chr7A_30892530_T / C,Chr7A_30892636_G / A,Chr7A_30893081_A / C,Chr7A_30893248_C / T,Chr7A_30893264_G / A,Chr7A_30894905_G / A,Chr7A_30895345_T / A,Chr7A_30895352_A / T,Chr7A_30895364_G / T,Chr7A_30895396_A / G,Chr7A_30895413_G / A,Chr7A_30895477_A / G,Chr7A_30895626_C / T,Chr7A_30898570_G / T,Chr7A_30900735_C / T,Chr7A_30900980_A / G,Chr7A_30901058_A / T,Chr7A_30903866_G / A,Chr7A_30904344_G / A, Chr7A_30914244_G / A,Chr7A_30925635_C / T,Chr7A_30925640_C / T,Chr7A_30925659_A / T,Chr7A_30932762_A / T,Chr7A_30940974_A / G,Chr7A_31143985_G / A,Chr7A_31144853_C / T,Chr7A_31145673_C / T,Chr7A_31145768_T / C,Chr7A_31146593_C / T,Chr7A_31147976_G / A,Chr7A_31147977_T / C, Chr7A_31148501_A / G,Chr7A_31160834_A / T,Chr7A_31161521_C / T,Chr7A_31161548_C / A,Chr7A_31161554_G / A,Chr7A_31170545_C / A,Chr7A_31170932_T / A,Chr7A_31171449_C / T,Chr7A_31220612_T / A,Chr7A_31220624_A / G,Chr7A_31234283_G / A,Chr7A_31384339_T / A,Chr7A_31420434_A / G, Chr7A_31420833_T / C,Chr7A_31421014_G / A,Chr7A_31421016_T / C,Chr7A_31465333_T / C,Chr7A_31465343_C / T,Chr7A_31465350_G / A,Chr7A_31465491_C / T,Chr7A_31465992_C / T,Chr7A_31465993_C / T,Chr7A_31465995_G / A,Chr7A_31466001_A / T,Chr7A_31466421_G / T,Chr7A_31479815_C / T,Chr7A_31479831_C / T,Chr7A_31479840_T / C,Chr7A_31479897_C / T,Chr7A_31479928_A / G,Chr7A_31479984_C / A,Chr7A_31482728_A / C,Chr7A_31485022_A / G,Chr7A_31485027_A / G,Chr7A_31485052_A / T,Chr7A_31485151_A / T,Chr7A_31488313_C / G,Chr7A_3151 6628_G / A,Chr7A_31518809_G / A,Chr7A_31519031_C / T,Chr7A_31519114_A / T,Chr7A_31520548_C / A,Chr7A_31521077_C / T,Chr7A_31521099_T / A,Chr7A_31521277_C / T,Chr7A_31521340_C / T,Chr7A_31521506_G / A,Chr7A_31521595_G / A,Chr7A_31521758_C / T,C hr7A_31521870_C / T,Chr7A_31521894_C / T,Chr7A_31524952_T / C,Chr7A_31532475_G / T,Chr7A_31537394_C / T,Chr7A_31556841_A / G,Chr7A_31876085_G / C,Chr7A_31877224_T / C,Chr7A_31877301_T / A,Chr7A_31877424_C / T,Chr7A_31877728_G / A,Chr7A_31900 559_G / A,Chr7A_31901028_T / A,Chr7A_32547445_T / C,Chr7A_34436369_A / G,Chr7A_35368267_C / T,Chr7A_35768998_G / A,Chr7A_43490348_C / G,Chr7A_47206152_G / A,Chr7A_47315753_C / T,Chr7A_48836095_C / T,Chr7A_56946307_G / T,Chr7A_59075352_T / C,Ch r7A_67604192_C / T,Chr7A_67604193_A / G,Chr7B_177532_A / G,Chr7B_1916898_C / G,Chr7B_3230059_G / T,Chr7B_3230074_A / G,Chr7B_3990844_T / C,Chr7B_6603491_G / T,Chr7B_7284354_G / A,Chr7B_26453266_G / A,Chr7B_26453325_A / G,Chr7B_26453379_T / C,C hr7B_26453397_C / T,Chr7B_26453399_G / A,Chr7B_26474514_G / A,Chr7B_31622066_C / T,Chr7B_31623393_T / C,Chr7B_46513292_T / G,

[0077] SNP sites were determined through genome-wide association analysis of 358 rose resequencing materials, and were screened using multiple software and models.

[0078] Example 2

[0079] The present invention provides a probe for predicting the vase life of roses. The probe is a liquid-phase probe whose sequence is designed based on the SNP site. It is used to hybridize with the genomic DNA of roses, capture DNA fragments in the region where the target SNP site is located, and perform subsequent sequencing analysis to determine the genotype of the rose to be tested, thereby predicting its vase life trait.

[0080] Example 3

[0081] The present invention provides a prediction chip for the vase life of roses. The prediction chip includes probes for detecting the 1679 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, the accurate prediction of the vase life trait of roses can be achieved.

[0082] Example 4

[0083] The present invention relates to the application of a combination of SNP molecular markers, probes, or chips in the prediction of the vase life of roses, by extracting genomic DNA from rose plants and using the SNP site combination, probes, or chips for detection.

[0084] Example 5

[0085] The present invention provides a method for predicting the vase life of roses based on SNP molecular marker combinations, comprising the following steps:

[0086] (1) Extract genomic DNA from the rose plants to be tested;

[0087] (2) Constructing a library of genomic DNA;

[0088] (3) Use probes to hybridize and capture DNA fragments in the library to enrich DNA fragments containing target SNP sites;

[0089] (4) Perform high-throughput sequencing on the captured DNA fragments;

[0090] (5) Compare and analyze the sequencing data with the rose reference genome to determine the genotype of the SNP site;

[0091] (6) Input the genotype data of the SNP loci into the whole genome selection model to obtain the prediction results of the vase life trait of the rose to be tested.

[0092] Example 6

[0093] The present invention relates to the application of an SNP site combination, probe, chip, or prediction method in rose molecular breeding, which is used to predict and screen the vase life trait of breeding materials in an early, accurate, and efficient manner during the rose breeding process, thereby shortening the breeding cycle, improving selection efficiency, and cultivating new rose varieties with excellent vase life traits.

[0094] A genome-wide selection model based on SNP site combinations can predict the vase life of roses with an accuracy greater than 0.710.

[0095] Example 7

[0096] The present invention provides a molecular breeding method for roses based on SNP markers, comprising the following steps:

[0097] (1) Extracting genomic DNA from roses;

[0098] (2) Use the above-mentioned liquid phase breeding chip to capture and enrich genomic DNA;

[0099] (3) Perform high-throughput sequencing on the captured and enriched DNA;

[0100] (4) Analyze the sequencing data and detect the genotypes of the 1679 SNP sites.

[0101] Example 8

[0102] A genome-wide association analysis of bottle life was performed on 358 rose whole-genome resequencing data, including the following workflow:

[0103] (1) Genomic DNA was extracted from 358 rose leaf tissues 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, quantified by qbit, and then sequenced using BGI DNBSEQ-T7.

[0104] (2) The second-generation sequencing data in fq.gz format obtained from step (1) was compared with the R. hybrida 'Samantha' genome (v2) using the bwa software.

[0105] (https: / / figshare.com / articles / dataset / _i_R_hybrida_i_Samantha_genome_v2_ / 28738781?file=53454104) Genome. SNP variation data were analyzed using GATK software, ultimately identifying 115,219,112 high-quality SNP loci.

[0106] (3) The SNP sites selected in step (2) were filtered to retain SNP sites with allele frequencies greater than 0.01 and deletion rates less than 0.2, resulting in 35,335,433 high-quality SNP sites. The population allele frequencies ( Figure 1 ), missing rate ( Figure 2 and heterozygosity ( Figure 3 The distribution meets the requirements for subsequent correlation analysis.

[0107] (4) Collection of agronomical traits related to vase life. Healthy, disease-free flower stems of similar developmental stage were selected. In a standardized environment, the depth of the flower stems in the vase was fixed at 10-15 cm, and the water was changed every 2-3 days. Vase life refers to the duration from when the flower stems were placed in water until the flowers exhibit obvious signs of decay (such as severe fading, wilting, and falling off of petals, and drying of stamens), losing their ornamental value. The unit is days. Three replicate vase life data points were collected, and the mean and BLUP values ​​were calculated. A total of five data points were used for GWAS correlation analysis.

[0108] (5) Using emmax, gemma, rmvp (FARMCPU model, GLM model, MLM model), and tassel (GLM model), six models were used to associate the agronomical trait of bottle life. The p-value threshold was set to 1E-4, and 268,779 SNP markers were definitely associated. After deduplication of the association results from different software models, 134,868 SNP sites were obtained.

[0109] (6) Probe evaluation of the SNPs screened in step (5). Screening criteria: no InDel markers within 50 bp upstream and downstream of the SNP site; 100 bp upstream and downstream of the sequence, with only one copy number in the whole genome; GC content of 40-60%; no short repetitive sequences in the sequence; no N bases in the sequence, resulting in 5710 SNP sites remaining.

[0110] (7) The SNP loci selected in step (6) are sorted according to the number of software and models associated and the association results. The 1679 SNP markers with the most software associations and the most significant association values ​​are selected. Finally, 1679 SNP markers are selected (see Table 1 for details). Their chromosome distribution characteristics are as follows: Figure 4 As shown. Figure 4 As shown, Figure 4 The whole chromosome marker distribution map of the vase life of roses provided by this invention.

[0111] (8) The SNP loci selected in step (7) were evaluated using 13 genome-wide selection models: LightGBM (lgb), Random Forest (RF), SVR (svr), Lasso Regression (Lasso), Ridge Regression (Ridge), rrBLUP (rrblup), Reproducing Kernel Hilbert Space (RKHS), Bayes Ridge Regression (BRR), Bayesian Lasso (BL), BayesC, BayesB, BayesA, and GBLUP. The genotypes and agronomical traits of 348 accessions were used to assess the accuracy of vase life prediction. The results showed that Ridge had the lowest prediction accuracy, while Bayes Ridge Regression (BRR) had the highest. The overall prediction accuracy was greater than 0.710, indicating good prediction accuracy. Figure 5 As shown, Figure 5 The diagram shows the genome-wide selection evaluation results of the vase life of roses provided by this invention.

[0112] (9) The SNP sites selected in step (7) are used to synthesize probe sequences for capture sequencing. The prediction accuracy of each model is as follows: Figure 5 As shown, the Bayes Ridge Regression (BRR) model has the highest prediction accuracy, with an overall accuracy greater than 0.710.

[0113] Example 9

[0114] The method for detecting roses using a liquid phase probe corresponding to 1679 SNPs as described in Example 8

[0115] (1) Genomic DNA extraction: Rose leaves were collected, stored with ice packs, and transported back to the laboratory in a timely manner. Total DNA of the rose was extracted using the magnetic bead method.

[0116] (2) Genomic DNA library construction: Genomic DNA fragmentation (200-300bp), DNA fragment end repair and adapter ligation, Pre-PCR amplification library.

[0117] (3) Probe hybridization with target region: The prepared SNP probe was hybridized with the Pre-PCR library. The streptomycin-modified probe was complementary to the complementary DNA library for capture. After enriching the target DNA library, other library DNA was eluted, resulting in the captured Pre-PCR library. Hybridization conditions: The streptomycin-labeled probe was hybridized with the target library at 50℃ for 16 h. Biotin-labeled magnetic beads were used to bind with the streptomycin-labeled probe at room temperature for 30 min to enrich the target library. After rinsing the non-target region library with wash buffer, it was eluted with Nuclease-free Water.

[0118] (4) After the magnetic beads are purified, they are amplified and enriched to the DNA concentration required for high-throughput sequencing, and then high-throughput sequencing is performed.

[0119] (5) High-throughput sequencing: The library obtained after capture and amplification in step (4) is subjected to high-throughput sequencing using the BGI Genomics DNBSEQ-T7 sequencing platform to obtain the sequencing results of genomic DNA, and the obtained data is cleaned.

[0120] (6) Liquid phase probe capture rate evaluation: The cleaned sequencing data were aligned to the R. hybrida 'Samantha' reference sequence using bwa software to evaluate its capture efficiency.

[0121] Experimental Example 1

[0122] Evaluation of liquid phase probe capture efficiency for 12 seasonal varieties

[0123] Experimental materials: Twelve rose leaf samples (covering 11 varieties, including two independent individuals from the Circus variety) were selected, and specific information is shown in Table 2. Samples were immediately flash-frozen in liquid nitrogen after collection and stored at -80℃ for later use. Genomic DNA was extracted from each individual plant using the CTAB method. Agarose gel electrophoresis and Nanodrop analysis showed that the OD260 / 280 ratios were both between 1.8 and 2.0, with a concentration ≥50 ng / μL, meeting the requirements for library construction.

[0124] Table 2

[0125] Test1 Morris Test2 Coffee Dream Test3 Gold bulls Test4 Black Buck Test5 circus Test6 circus Test7 Teng Tianmeng Test8 Chantal Test9 Colorful Orange Bowl Test10 Ingrid Bergman Test11 Iver's Rose Test12 Couples dating

[0126] Experimental methods

[0127] (1) Library Construction

[0128] DNA fragments were broken down to 200-300 bp using a Covaris M220 ultrasonic disruptor.

[0129] The KAPAHyperPrep library preparation kit is used for end repair, adapter addition, and pre-amplification.

[0130] (2) Target area capture

[0131] The liquid-phase probe with 1679 SNP sites (NimbleGen SeqCap EZ system) designed in Example 8 was used.

[0132] Denaturation at 95℃ for 5 min, hybridization at 65℃ for 16 h, and enrichment of target fragments with streptavidin magnetic beads.

[0133] (3) High-throughput sequencing

[0134] BGI DNBSEQ-T7 platform, PE150 sequencing mode;

[0135] Average data size: 1.5 Gb / sample (range: 1.2-1.8 Gb).

[0136] (4) Data Analysis

[0137] Coverage depth: SAMtools calculates the average depth of the target SNP region;

[0138] Detection rate: The proportion of successfully genotyped SNPs to the total number of markers (threshold: depth ≥30X, base quality Q ≥30).

[0139] 3. Capture efficiency evaluation results

[0140] Results analysis:

[0141] Ultra-high coverage depth: The average depth of all samples was >390X (range: 393.67-530.84X), far exceeding the minimum detection threshold (30X); the highest depth sample was Iver's Rose (530.84X), and the lowest was Morris (393.67X). Stable high detection rate: The detection rate ranged from 97.62% to 98.81% (mean 98.24% ± 0.39%); there were small differences between individuals of the same variety (e.g., Circus individual 1: 98.81% vs. individual 2: 97.66%). Key conclusions: The capture efficiency of 1679 SNP markers remained stable between 97.62% and 98.81%, with coverage depths all exceeding 390X, demonstrating that the liquid-phase probe design can accurately cover genomic variations across multiple rose varieties, meeting the requirements for genotyping accuracy in whole-genome selection breeding.

[0142] Table 3

[0143]

[0144] It provides technical support; it is significantly superior to traditional solid-phase chips (average detection rate <95%), and is suitable for large-scale breeding screening.

[0145] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope. The scope of protection of the present invention is defined by the appended claims, specification, and their equivalents.

Claims

1. The application of an SNP molecular marker combination in molecular breeding of rose vase life, characterized in that: The SNP molecular marker combination consists of 1679 SNP loci distributed on the 14 chromosomes of the rose. The specific information of the SNP loci is represented in the structure of chromosome number_physical location_allele genotype. The specific locus information is shown in Table 1 of the specification. The reference genome for the SNP molecular marker combination is R. hybrida 'Samantha' genome v2 version.

2. A molecular breeding method for vase life of roses based on the SNP molecular marker combination described in claim 1, characterized in that... Includes the following steps: (1) Extracting genomic DNA from roses; (2) Using the liquid-phase breeding chip with SNP molecular marker combination as described in claim 1 to capture and enrich genomic DNA; (3) Perform high-throughput sequencing on the captured and enriched DNA; (4) Analyze the sequencing data and detect the genotypes of the 1679 SNP sites.

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