Chinese rose petal outer edge wave whole genome SNP molecular marker combination and application thereof

Abstract

The invention discloses a Chinese rose petal outer edge wave whole genome SNP (Single Nucleotide Polymorphism) molecular marker combination and application thereof, and aims to solve the problems that the traditional Chinese rose breeding period is long, the Chinese rose breeding is easily interfered by the environment, and the petal wave character cannot be predicted in a seedling or seed stage. According to the method, 358 Chinese rose whole genome re-sequencing data and petal outer edge wave phenotype association analysis are integrated, a multi-model combined screening strategy is adopted, 1453 SNP markers obviously associated with petal outer edge waves are determined, and a whole genome selection model with high prediction precision is constructed. By combining a specific probe and chip technology, high-throughput and rapid detection is realized, the detection period can be shortened, the petal wave traits can be directly predicted by utilizing genotype data in a seedling stage, the breeding process is accelerated, and core data support is provided for Chinese rose petal outer edge wave genetic law analysis and molecular marker-assisted breeding; the method has outstanding theoretical value and application potential.

Description

Technical Field

[0001] The present invention relates to the field of molecular breeding technology, and in particular to a whole-genome SNP molecular marker combination for rose petal outer edge waves and an application thereof. Background Art

[0002] The wavy outer edge of rose petals is a key aesthetic feature of ornamental flowers, directly impacting their market value and breeding objectives. Traditional breeding relies primarily on phenotypic screening, requiring multiple generations of field trials to observe the genetic stability of traits. This is not only time-consuming, typically taking 3-5 years, but also highly susceptible to interference from environmental factors such as light and temperature, resulting in inefficient trait improvement.

[0003] In recent years, genome-wide association studies (GWAS) and genome-wide selection (GS) have provided new avenues for marker-assisted breeding. Single nucleotide polymorphisms (SNPs), as high-density molecular markers, have been widely used for genetic analysis of macroscopic traits such as flower shape and color. However, for complex morphological features such as the wavy outer edge of petals, current research remains limited.

[0004] Existing technologies often rely on single-model association analysis to screen for SNPs, resulting in high redundancy and insufficient specificity in marker combinations. Furthermore, the lack of genome-wide selection models suitable for complex phenotypes limits prediction accuracy and practicality. Furthermore, the lack of mature probes and microarray technologies targeting petal wave traits severely restricts the application of molecular markers in early breeding.

[0005] Existing technologies have obvious deficiencies in molecular marker-assisted breeding of the wavy trait on the outer edge of rose petals. It is urgent to develop a whole-genome SNP molecular marker combination for the wavy trait on the outer edge of rose petals that can efficiently and accurately predict this trait and its application, so as to overcome the limitations of traditional breeding methods and improve breeding efficiency and accuracy. Summary of the Invention

[0006] In order to solve the problems of the prior art, the purpose of the present invention is to provide a genome-wide SNP molecular marker combination for the outer edge wave of rose petals and its application.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present application provides a genome-wide SNP molecular marker combination for the outer edge waves of rose petals.

[0009] In a second aspect, the present application provides a probe for predicting the waves on the outer edge of rose petals.

[0010] In a third aspect, the present application provides a chip for predicting the waves on the outer edge of rose petals.

[0011] In a fourth aspect, the present application provides an application of a SNP molecular marker combination, probe or chip in predicting the outer edge waves of rose petals.

[0012] In a fifth aspect, the present application provides a method for predicting the outer edge waves of rose petals based on a combination of SNP molecular markers.

[0013] In a sixth aspect, the present application provides an application of a SNP site combination, probe, chip or prediction method in rose molecular breeding.

[0014] In a seventh aspect, the present application provides a rose molecular breeding method based on SNP markers.

[0015] The first aspect of the present application provides a genome-wide SNP molecular marker combination for the wavy outer edge of rose petals, which includes 1,453 SNP sites distributed on the 14 chromosomes of rose; the specific information of the SNP site is represented by a structure of chromosome number_physical position_allele type, and the SNP site contains a dominant allele that is positively and significantly associated with the wavy outer edge of rose petals, and the specific site information is shown in Table 1.

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

[0017] The second aspect of the present application provides a prediction probe for the waves on the outer edge of rose petals. The screening principles of the probe include: it is designed from a combination of SNP sites, can specifically bind to the SNP sites, is used to capture the target DNA region, and realizes the detection of specific SNP sites. Its sequence information is associated with the SNP site combination, and complies with the various screening principles of probe design, including no Indel markers within 50bp upstream and downstream of the SNP site, only one copy number of the 100bp upstream and downstream sequences in the entire genome, a GC content of 40-60%, no short fragment repeat sequences in the sequence, and no N bases.

[0018] The third aspect of the present application provides a prediction chip for the wavy outer edge of rose petals. The prediction chip includes probes for detecting the 1453 SNP sites. By detecting the genotype of the 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 wavy traits of the outer edge of rose petals can be achieved.

[0019] The fourth aspect of the present application provides an application of a SNP molecular marker combination, probe or chip in predicting the waves on the outer edge of rose petals, by extracting genomic DNA from rose plants and using SNP site combinations, probes or chips for detection.

[0020] A fifth aspect of the present application provides a method for predicting rose petal outer edge waves based on a combination of SNP molecular markers, comprising the following steps:

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

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

[0023] (3) using probes to perform hybridization capture on the DNA fragments in the library to enrich the DNA fragments containing the target SNP site;

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

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

[0026] (6) The genotype data of the SNP locus combination are input into the whole genome selection model to obtain the prediction results of the wavy traits of the outer edge of the petals of the tested rose.

[0027] Furthermore, in step (3), the hybridization reaction system includes the following components: 10-20 μL of Pre-PCR amplification library; 20-30 μL of liquid probe mixture, wherein the concentration of each liquid probe in the liquid probe mixture is 50-100 nM; 5-10 μL of hybridization buffer, wherein the hybridization buffer contains 3-5 mol / L salt solution and 1%-2% blocking agent; and RNase-free water is added to make up the reaction system to 50-100 μL.

[0028] 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 wavy traits of the petal edges of breeding materials during the rose breeding process, thereby shortening the breeding cycle, improving the selection efficiency, and cultivating new rose varieties with excellent petal edge wavy traits.

[0029] Furthermore, the whole-genome selection model constructed based on the SNP site combination can predict the wavy outer edge of rose petals with a prediction accuracy greater than 0.563.

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

[0031] (1) Collect rose samples and extract genomic DNA: Leaf samples of multiple rose varieties were collected, and total DNA was extracted using the magnetic bead method. After fragmentation and end repair, sequencing adapters were connected to construct a pre-PCR amplification library;

[0032] (2) SNP marker detection: A liquid-phase breeding chip containing specific probes is used to hybridize and capture the DNA in the sample library, specifically enrich the target SNP region DNA, and obtain the captured library after elution and purification; the captured library is subjected to high-throughput sequencing on the BGI DNBSEQ-T7 platform, and after quality control, it is aligned to the rose reference genome to determine the sample genotype data. The SNP markers are 1453 SNP markers across 14 chromosomes that are significantly associated with the petal outer edge wave, which are determined by integrating 358 rose whole genome resequencing data and systematic petal outer edge wave phenotype association analysis using a multi-model joint screening strategy;

[0033] (3) Phenotypic data collection: The phenotypic data of the outer edge of rose petals were collected, and three repeated observations were recorded according to the standardized scoring system. After calculating the BLUP value, it was combined with five types of phenotypic indicators for subsequent association analysis;

[0034] (4) Trait prediction based on whole genome selection model: Based on the sample genotype data and phenotypic data, the whole genome selection model was trained using algorithms such as LightGBM, random forest, and Bayesian ridge regression. The model was used to predict the wavy traits of the petal outer edge of the breeding material and select candidate individuals with excellent wavy traits. Among them, the Bayesian ridge regression prediction accuracy was the best (R 2 >0.563);

[0035] (5) Breeding decision-making: Based on the prediction results, select candidate individuals with excellent petal outer edge wavy traits for reproduction and breeding to accelerate the rose molecular breeding process.

[0036] Beneficial effects: The present invention combines specific probes with chip technology to achieve high-throughput and rapid detection, which can shorten the detection cycle and use genotype data to directly predict the wavy traits of petals in the seedling stage, accelerate the breeding process, and provide core data support for the analysis of the genetic laws of the wavy outer edge of rose petals and molecular marker-assisted breeding. It has outstanding theoretical value and application potential.

[0037] Compared with the prior art, the present invention has the following advantages:

[0038] (1) Improved breeding efficiency: Traditional breeding methods rely on manual observation of the wavy petal phenotype, requiring multiple generations of field planting and phenotypic recording, which is a long process, typically 3-5 years. However, this invention develops a combination of SNP markers highly associated with the wavy petal trait, combines specific probes with chip technology, and achieves high-throughput, rapid detection, shortening the detection cycle and accelerating the breeding process.

[0039] (2) High prediction accuracy: Existing technologies cannot predict petal wave traits at the seedling or seed stage, and screening must wait until the plant blooms. The present invention is based on a whole genome selection model and uses the genotype data of seedlings to directly predict petal wave traits without waiting for the plant to bloom. The SNP marker combination is determined through a multi-model joint screening strategy, and a variety of machine learning algorithms are used to train the model. The prediction accuracy of the Bayesian Ridge regression model is R 2 >0.563, significantly better than the traditional linear model.

[0040] (3) Improved detection efficiency: The probe and chip technology for the petal wave trait is not yet mature, which restricts the application of molecular markers in early breeding. The SNP marker combination of the present invention covers 14 chromosomes. Combining specific probes and chip technology, it achieves a target site capture efficiency of ≥98% and a coverage depth of ≥50×. It can efficiently perform large-scale sample detection and provide core data support for the analysis of the genetic pattern of the outer edge wave of rose petals and molecular marker-assisted breeding.

[0041] (4) Reduce costs: By performing genotyping tests at the seedling or seed stage to predict the petal wave trait, the manpower, material resources, and time costs required for multi-generation field planting and phenotypic recording in traditional breeding are avoided, thereby reducing breeding costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] 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.

[0043] Figure 1 The present invention provides a full chromosome marker distribution map of the wavy outer edge of rose petals.

[0044] Figure 2 This is the allele frequency diagram of the wavy outer edge of rose petals provided by the present invention.

[0045] Figure 3 This is a graph showing the missing rate of waves on the outer edge of rose petals provided by the present invention.

[0046] Figure 4 This is a heterozygosity diagram of the wavy outer edge of rose petals provided by the present invention.

[0047] Figure 5 This is a diagram of the genome-wide selection evaluation results of the wavy outer edge of rose petals provided by the present invention. DETAILED DESCRIPTION

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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 size 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.

[0055] In a first aspect of an embodiment of the present application, a whole-genome SNP molecular marker combination for the wavy outer edge of rose petals is provided. The SNP molecular marker combination includes 1,453 SNP sites distributed on the 14 chromosomes of rose. The specific information of the SNP site is represented by a structure of chromosome number_physical position_allele type. The SNP site includes a dominant allele that is positively and significantly associated with the wavy outer edge of rose petals. The specific site information is shown in Table 1.

[0056] The SNP sites were determined through genome-wide association analysis of 358 rose resequencing materials and association screening using multiple software and models.

[0057] The second aspect of the embodiment of the present application provides a prediction probe for the waves on the outer edge of rose petals. The screening principles of the probe include: it is designed from a combination of SNP sites, can specifically bind to the SNP site, is used to capture the target DNA region, and realizes the detection of specific SNP sites. Its sequence information is associated with the SNP site combination, and complies with various screening principles of probe design, including no Indel markers within 50bp upstream and downstream of the SNP site, only one copy number of the 100bp upstream and downstream sequences in the entire genome, a GC content of 40-60%, no short fragment repeat sequences in the sequence, and no N bases.

[0058] The third aspect of an embodiment of the present application provides a prediction chip for the wavy outer edge of rose petals. The prediction chip includes probes for detecting the 1453 SNP sites. By detecting the genotype of the SNP sites in the genomic DNA of the rose sample to be tested and utilizing a pre-constructed whole-genome selection model, accurate prediction of the wavy traits of the outer edge of rose petals can be achieved.

[0059] The fourth aspect of the embodiments of the present application provides an application of a SNP molecular marker combination, probe or chip in predicting the waves on the outer edge of rose petals, by extracting genomic DNA from rose plants and using the SNP site combination, probe or chip for detection.

[0060] A fifth aspect of the present application provides a method for predicting rose petal outer edge wavy based on a combination of SNP molecular markers, comprising the following steps:

[0061] (1) Extracting genomic DNA from the rose plants to be tested;

[0062] (2) constructing a library of the genomic DNA;

[0063] (3) using probes to perform hybridization capture on the DNA fragments in the library to enrich the DNA fragments containing the target SNP site;

[0064] (4) performing high-throughput sequencing on the captured DNA fragments;

[0065] (5) Compare the sequencing data with the rose reference genome to determine the genotype of the SNP site;

[0066] (6) The genotype data of the SNP locus combination are input into the whole genome selection model to obtain the prediction results of the wavy traits of the outer edge of the petals of the tested rose.

[0067] In some embodiments, in step (3), the hybridization reaction system includes the following components: 10-20 μL of Pre-PCR amplification library; 20-30 μL of liquid probe mixture, wherein the concentration of each liquid probe in the liquid probe mixture is 50-100 nM; 5-10 μL of hybridization buffer, wherein the hybridization buffer contains 3-5 mol / L salt solution and 1%-2% blocking agent; RNase-free water is added to make up the reaction system to 50-100 μL.

[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 wavy traits of the petal edges of breeding materials during the rose breeding process, thereby shortening the breeding cycle, improving the selection efficiency, and cultivating new rose varieties with excellent petal edge wavy traits.

[0069] In some embodiments, in some embodiments, the whole genome selection model constructed based on the combination of SNP sites can predict the outer edge waves of rose petals with a prediction accuracy greater than 0.563.

[0070] A seventh aspect of the present invention provides a rose molecular breeding method based on SNP markers, comprising the following steps:

[0071] (1) Collect rose samples and extract genomic DNA: Leaf samples of multiple rose varieties were collected, and total DNA was extracted using the magnetic bead method. After fragmentation and end repair, sequencing adapters were connected to construct a pre-PCR amplification library;

[0072] (2) SNP marker detection: A liquid-phase breeding chip containing specific probes is used to hybridize and capture the DNA in the sample library, specifically enrich the target SNP region DNA, and obtain the captured library after elution and purification; the captured library is subjected to high-throughput sequencing on the BGI DNBSEQ-T7 platform, and after quality control, it is aligned to the rose reference genome to determine the sample genotype data. The SNP markers are 1453 SNP markers across 14 chromosomes that are significantly associated with the petal outer edge wave, which are determined by integrating 358 rose whole genome resequencing data and systematic petal outer edge wave phenotype association analysis using a multi-model joint screening strategy;

[0073] (3) Phenotypic data collection: The phenotypic data of the outer edge of rose petals were collected, and three repeated observations were recorded according to the standardized scoring system. After calculating the BLUP value, it was combined with five types of phenotypic indicators for subsequent association analysis;

[0074] (4) Trait prediction based on whole genome selection model: Based on the sample genotype data and phenotypic data, the whole genome selection model was trained using algorithms such as LightGBM, random forest, and Bayesian ridge regression. The model was used to predict the wavy traits of the petal outer edge of the breeding material and select candidate individuals with excellent wavy traits. Among them, the Bayesian ridge regression prediction accuracy was the best (R 2 >0.563);

[0075] (5) Breeding decision-making: Based on the prediction results, select candidate individuals with excellent petal outer edge wavy traits for reproduction and breeding to accelerate the rose molecular breeding process.

[0076] Example 1

[0077] The present invention discloses a genome-wide SNP molecular marker combination for the outer edge wavy pattern of rose petals. The SNP molecular marker combination includes 1453 SNP sites distributed on 14 rose chromosomes. The specific information of the SNP sites is represented by a structure of chromosome number_physical position_allele type. The SNP sites include a dominant allele that is significantly positively associated with the outer edge wavy pattern of rose petals. The specific site information is shown in Table 1.

[0078] Table 1

[0079] Chr1A_425134_T / C,Chr1A_15929905_C / T,Chr1A_19411802_C / T,Chr1A_26955141_A / C,Chr1A_30699107_C / T,Chr1A_30703342_T / C,Chr1A_30703412_T / C,Chr1A_30703427_T / A,Chr1A_30705914_T / G,Chr1A_30855826_A / G,Chr1A_31083707_G / A,Chr1A_32219641_G / T,Chr1A_32258985_T / A,Chr1A_32808681_T / G,Chr1A_32808835_C / T,Chr1A_32812951_C / T,Chr1A_32868129_A / G,Chr1A_32873223_A / T,Chr1A_34014639_G / A,Chr1A_34948288_G / A,Chr1A_34988619_G / C,Chr1A_35005875_G / A,Chr1A_35005940_G / A,Chr1A_35041151_T / A,Chr1A_35041190_C / T,Chr1A_35664718_T / A,Chr1A_40664065_G / A,Chr1B_5664230_A / T,Chr1B_5831630_G / A,Chr1B_6589081_C / A,Chr1B_15336459_G / C,Chr1B_15336790_C / T,Chr1B_15336791_A / T,Chr1B_23029619_T / C,Chr1B_25029950_C / T,Chr1B_48397304_G / T,Chr2A_119108_T / G,Chr2A_7668086_C / T,Chr2A_15417427_C / G,Chr2A_15417907_G / A,Chr2A_15420521_T / C,Chr2A_15420530_C / T,Chr2A_15427044_G / A,Chr2A_15428131_A / G,Chr2A_15428358_A / G,Chr2A_15428360_C / T,Chr2A_15428790_A / C,Chr2A_24927180_G / A,Ch r2A_25405386_G / T,Chr2A_35112069_C / T,Chr2A_47390738_G / A,Chr2A_49547237_G / A,Chr2A_60064414_T / C,Chr2A_60064434_G / C,Chr2A_60064447_T / C,Chr2A_60064486_A / G,Chr2A_60064502_C / T,Chr2A_60064512_A / C,Chr2A_60064524_C / T,Chr2A_60064528_C / T,Chr2B_58685_C / T,Chr2B_25918995_G / A,Chr2B_25919053_T / G,Chr2B_25919088_C / T,Chr2B_25919203_A / G,Chr2B_25921765_C / T,Chr2B_25921792_C / T,Chr2B_25921813_G / A,Chr2B_25921822_G / A,Chr2B_25921864_A / G,Chr2B_25922377_G / A,Chr2B_25923702_T / A,Chr2B_47333573_A / T,Chr2B_50770920_A / T,Chr2B_50770947_C / T,Chr2B_50770984_A / G,Chr2B_50771039_A / T,Chr2B_84258981_G / C,Chr3A_7956672_C / T,Chr3A_25657525_T / G,Chr3A_33682876_T / C,Chr3A_47667612_G / T,Chr3B_8347428_A / G,Chr3B_27015014_T / C,Chr3B_27015046_A / G,Chr3B_27015071_G / A,Chr3B_27015280_C / T,Chr3B_27015338_C / T,Chr3B_27392279_G / A,Chr3B_27392284_A / C,Chr3B_27392460_T / A,Chr3B_27392562_A / G,Chr3B_27392610_G / A,Chr3B_27392658_T / C,Chr3B_27392763_A / G,Chr3B_27392805_C / T,Chr3B_27392859_T / G,Chr3B_27393194_C / A,Chr3B_27393231_A / G,Chr3B_27393290_G / T,Chr3B_27393318_C / T,Chr3B_27393354_A / G,Chr3B_27393511_A / G,Chr3B_27393524_C / T,Chr3B_27393595_G / C,Chr3B_27393627_C / T,Chr3B_27393658_T / A,Chr3B_27393670_A / T,Chr3B_27393732_T / A,Chr3B_27393735_G / A,Chr3B_27394785_T / A,Chr3B_27394790_T / C,Chr3B_27394793_C / A,Chr3B_27394968_T / C,Chr3B_27395019_A / T,Chr3B_27395047_C / T,Chr3B_27395061_G / A,Chr3B_27395103_A / G,Chr3B_27395167_C / T,Chr3B_27395817_G / T,Chr3B_27395828_A / C,Chr3B_27395844_T / C,Chr3B_27395867_G / A,Chr3B_27395894_G / T,Chr3B_27395985_C / A,Chr3B_27396108_A / C,Chr3B_27396153_C / T,Chr3B_27484863_C / G,Chr3B_28839552_C / T,Chr3B_43108829_C / T,Chr3B_44193020_T / A,Chr3B_44627899_A / C,Chr3B_44884476_T / C,Chr3B_44900715_A / G,Chr3B_44917902_T / C,Chr3B_44917917_C / T,Chr3B_44917964_C / A,Chr3B_44946883_T / C,Chr3B_44952518_C / T,Chr3B_44963797_C / T,Chr3B_44964443_G / T,Chr3B_44964572_G / T,Chr3B_44964618_C / T,Chr4A_10635811_G / A,Chr4A_11359114_G / T,Chr4A_11967861_A / G,Chr4A_11967872_G / T,Chr4A_11967948_T / G,Chr4A_11967965_A / G,Chr4A_14501491_A / G,Chr4A_15805683_G / A,Chr4A_16084796_C / A,Chr4A_16273354_T / G,Chr4A_16396167_G / A,Chr4A_16513239_T / C,Chr4A_16716575_T / G,Chr4A_16746579_T / C,Chr4A_16957638_G / C,Chr4A_16957646_C / A,Chr4A_16957690_C / T,Chr4A_17191671_T / A,Chr4A_17802830_A / C,Chr4A_19294259_T / A,Chr4A_20586575_T / C,Chr4A_32215338_C / T,Chr4A_32827433_T / C,Chr4A_42067885_C / T,Chr4A_45422689_C / T,Chr4A_62385760_T / C,Chr4A_62385766_C / T,Chr4B_4822513_C / A,Chr4B_6108377_G / A,Chr4B_6108423_G / T,Chr4B_6108431_C / A,Chr4B_6108432_A / C,Chr4B_9269733_G / T,Chr4B_9672064_T / C,Chr4B_9672080_T / C,Chr4B_9672172_C / A,Chr4B_9673536_A / G,Chr4B_9673548_C / T,Chr4B_10278298_A / T,Chr4B_12693349_T / G,Chr4B_13981199_A / C,Chr4B_14015562_C / G,Chr4B_14015592_G / A,Chr4B_14015664_A / G,Chr4B_14015671_C / T,Chr4B_14015686_G / 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C,Chr4B_21182683_A / T,Chr4B_21183309_C / G,Chr4B_21291057_A / T,Chr4B_21291074_T / G,Chr4B_21291200_T / C,Chr4B_21291373_A / G,Chr4B_21294852_A / G,Chr4B_21341632_C / A,Chr4B_21354564_C / T,Chr4B_21354580_C / T,Chr4B_21354636_A / C,Chr4B_21355044_A / T,Chr4B_21355050_A / G,Chr4B_21355071_C / A,Chr4B_21370502_T / C,Chr4B_21370834_T / C,Chr4B_21370838_G / A,Chr4B_21464395_C / T,Chr4B_21467651_T / G,Chr4B_21468012_C / T,Chr4B_21574525_A / G,C hr4B_21868718_T / C,Chr4B_21870205_T / G,Chr4B_21870229_T / C,Chr4B_21870403_A / G,Chr4B_21870682_A / G,Chr4B_21870973_A / T,Chr4B_21871041_A / G,Chr4B_21871188_A / T,Chr4B_21871244_A / G,Chr4B_21871260_A / G,Chr4B_21871271_G / A,Chr4B_21891047_T / C,Chr4B_21891054_C / T,Chr4B_21893713_C / T,Chr4B_21893744_A / G,Chr4B_21893747_T / G,Chr4B_21894088_T / A,Chr4B_21894391_C / T,Chr4B_21894395_A / C,Chr4B_21894401_T / C,Chr4B_22051264_T / C,Chr4B_22405200_A / G,Chr4B_22405296_A / T,Chr4B_22405369_T / A,Chr4B_22406281_A / G,Chr4B_22406334_T / C,Chr4B_22406496_T / G,Chr4B_22406506_T / C,Chr4B_22406889_A / G,Chr4B_22407013_C / G,Chr4B_22407299_T / C,Chr4B_22407394_G / C,Chr4B_22439535_A / G,Chr4B_22439628_G / A,Chr4B_22439708_G / A,Chr4B_22476152_T / C,Chr4B_22476327_G / A,Chr4B_22476482_A / G,Chr4B_22476623_T / C,Chr4B_22476848_G / A,Chr4B_22476873_G / T,Chr4B_22477731_A / G,Chr4B_22477829_G / T,C hr4B_22482504_T / C,Chr4B_22482515_A / G,Chr4B_22482604_A / G,Chr4B_22482695_A / G,Chr4B_22482720_C / A,Chr4B_22482735_T / C,Chr4B_22482744_A / G,Chr4B_22482759_T / C,Chr4B_22482797_T / A,Chr4B_22482820_A / G,Chr4B_22483103_C / T,Chr4B_22485787_T / C,Chr4B_22485847_T / C,Chr4B_22485854_G / A,Chr4B_22491303_A / G,Chr4B_22491362_A / G,Chr4B_22491411_A / G,Chr4B_22491428_A / G,Chr4B_22491549_T / C,Chr4B_22491577_A / G,Chr4B_22492003_T / C,Chr4B_22492492_A / G,Chr4B_22492646_T / C,Chr4B_22492682_A / G,Chr4B_22492699_C / T,Chr4B_22492710_G / C,Chr4B_22492731_A / C,Chr4B_24082050_A / G,Chr4B_24237284_A / C,Chr4B_24237324_G / A,Chr4B_24277635_T / C,Chr4B_24555563_C / G,Chr4B_24751958_A / G,Chr4B_24766478_T / C,Chr4B_24962329_A / G,Chr4B_28312949_C / A,Chr4B_28394363_A / G,Chr4B_28394428_A / C,Chr4B_28394480_C / A,Chr4B_28395363_T / C,Chr4B_28465373_T / C,Chr4B_28465375_G / A,Chr4B_28465383_C / T,Chr4B_28469446_C / T,Chr4B_28625184_G / C,Chr4B_28678213_T / G,Chr4B_28678257_T / G,Chr4B_28678263_A / T,Chr4B_28678271_T / C,Chr4B_28869100_A / G,Chr4B_30645777_G / A,Chr4B_30823999_C / A,Chr4B_30993432_C / T,Chr4B_30993538_G / T,Chr4B_30993540_A / G,Chr4B_31310805_T / G,Chr4B_31839630_A / G,Chr4B_31854083_A / C,Chr4B_31854085_G / T,Chr4B_33255142_A / G,Chr4B_37802591_G / A,Chr4B_52228571_C / T,Chr4B_56014691_T / G,Chr4B_56014702_C / T,Chr4B_56014740_T / C,Chr5A_4474624_C / T,Chr5A_5572372_A / C,C hr5A_5572475_A / G,Chr5A_5573139_C / G,Chr5A_5573163_C / A,Chr5A_5573175_C / A,Chr5A_5573262_C / A,C hr5A_5573284_C / T,Chr5A_5573618_G / A,Chr5A_15314372_A / C,Chr5A_20536441_A / G,Chr5A_21548081_T / A,Chr5A_23777019_A / T,Chr5A_28689471_A / G,Chr5A_28830705_G / A,Chr5A_31470437_C / T,Chr5A_31471814_C / A,Chr5A_49594481_C / T,Chr5A_49594531_C / T,Chr5A_49594573_G / A,Chr5A_55772434_T / C,Chr5A_55918738_C / T,Chr5A_55918739_G / A,Chr5A_56220939_G / A,Chr5A_56233111_G / A,Chr5A_56236363_A / G,Chr5A_56760609_G / T,Chr5A_56863212_G / C,Chr5A_56863216_G / A,Chr5A_56863894_A / G,Chr5A_57060688_T / C,Chr5A_59160085_T / C,Chr5A_59160111_C / T,Chr5A_59160347_T / G,Chr5A_59178082_C / T,Chr5A_59178716_T / C,Chr5A_59179137_A / C,Chr5A_59179537_A / C,Chr5A_59187072_T / A,Chr5A_59187779_G / A,Chr5A_59187821_G / A,Chr5A_59390736_C / G,Chr5A_67357198_G / A,Chr5A_74486766_G / A,Chr5A_74491526_G / A,Chr5A_79101524_A / T,Chr5A_81830117_C / T,Chr5A_82574573_G / A,Chr5A_825 74610_C / T,Chr5A_83322537_C / T,Chr5A_83325645_T / G,Chr5A_83690316_T / C,Chr5A_83993901_A / G,Chr5A_84111065_G / A,Chr5A_84728198_T / A, Chr5A_84728204_A / G,Chr5A_84853150_A / G,Chr5A_85550512_G / A,Chr5A_89986149_A / G,Chr5A_89986159_G / A,Chr5B_17867483_C / A,Chr5B_18005269_T / C,Chr5B_18005273_A / G,Chr5B_18005315_A / G,Chr5B_18018816_A / G,Chr5B_18020630_T / C,Chr5B_18041980_T / C,Chr5B_18645826_T / C, Chr5B_18653713_C / T,Chr5B_19249148_C / T,Chr5B_19318842_C / A,Chr5B_19318849_C / T,Chr5B_19936277_C / T,Chr5B_23671759_A / G,Chr5B_26562695_T / C,Chr5B_26562722_C / T,Chr5B_26812148_T / A,Chr5B_27063035_A / T,Chr5B_27075292_A / G,Chr5B_27075351_G / A,Chr5B_27075357_T / C, Chr5B_27075378_A / G,Chr5B_28244863_C / T,Chr5B_28245200_A / G,Chr5B_28245846_T / A,Chr5B_28246065_A / G,Chr5B_28246076_A / C,Chr5B_28248045_G / A,Chr5B_28248111_A / G,Chr5B_28248123_T / C,Chr5B_28251790_A / G,Chr5B_39007515_A / G,Chr5B_39007527_G / A,Chr5B_45133051_C / A,Chr5B_51909624_G / T,Chr5B_52602280_C / T,Chr5B_53125133_C / A,Chr5B_53392912_G / T,Chr5B_534 99884_G / A,Chr5B_53583002_G / A,Chr5B_53891198_T / A,Chr5B_54020991_T / C,Chr5B_54123161_T / G, Chr5B_54498167_G / T,Chr5B_54546886_A / G,Chr5B_54547433_A / G,Chr5B_54547445_C / G,Chr5B_54552441_T / C,Chr5B_54552447_C / T,Chr5B_54900248_G / A,Chr5B_54954629_A / G,Chr5B_54977775_T / A,C hr5B_54977880_T / C,Chr5B_54989523_A / C,Chr5B_54989748_T / A,Chr5B_54989925_C / G,Chr5B_54989958_C / T,Chr5B_54995906_C / A,Chr5B_54995968_T / A,Chr5B_54995992_C / T,Chr5B_54996000_A / T,Chr5B_55107907_G / A,Chr5B_55108076_C / T,Chr5B_55108192_A / C,C hr5B_55108225_C / A,Chr5B_55108771_A / G,Chr5B_55109159_C / T,Chr5B_57428540_C / T,Chr5B_57531487_G / T,Chr5B_57645639_G / A,Chr5B_57667977_A / G,Chr5B_59130143_T / G,Chr5B_61884706_G / T,Chr5B_65081825_A / G,Chr5B_65513011_A / C,Chr5B_65513158_T / C,C hr5B_65513159_T / C,Chr5B_65513200_C / T,Chr5B_66669524_C / T,Chr5B_72876075_A / G,Chr5B_72876114_T / C, Chr5B_72876188_G / C,Chr5B_77296730_C / T,Chr5B_77680545_G / C,Chr5B_77682541_G / A,Chr5B_77682543_G / A,Chr5B_79040211_A / G,Chr5B_79109109_T / C,Chr5B_79109114_C / A,Chr5B_79109498_T / C,Chr5B_79109503_C / T,Chr5B_79109526_A / G,Chr5B_80418574_G / A,Chr5B_81051952_C / T,Chr5B_81052020_A / G,Chr5B_81063036_T / C,Chr5B_81063058_A / T,Chr5B_81063061_T / C,Chr5B_82090616_A / T,Chr5B_82184886_T / C,Ch r5B_82184924_G / A,Chr5B_82710882_T / A,Chr5B_82711600_C / T,Chr5B_82711608_A / T,Chr5B_82711636_G / C,Chr5B_82711642_G / A,Chr5B_82780598_A / G,Chr5B_82881523_C / T,Chr5B_82882474_A / T,Chr5B_82882506_G / T,Chr5B_82917968_A / G,Chr5B_82918006_C / T,Chr5B_82918128_G / A,Chr5B_82918129_C / T,Chr5B_83891654_A / G,Chr5B_83891658_C / T,Chr5B_84979326_G / A,Chr5B_84979398_G / A,Chr5B_84979453_A / T,Chr5B_85929087_T / C,Chr6A_6734029_G / C,Chr6A_11679163_C / A,Chr6A_27160840_T / A,Chr6A_27918811_A / T,Ch r6A_27918877_C / T,Chr6A_28190145_C / T,Chr6A_37543281_A / C,Chr6A_37623280_A / T,Chr6A_37623310_A / T,Chr6A_37794833_G / T,Chr6A_37812716_T / A,Chr6A_39987799_G / T,Chr6A_40060471_G / A,Chr6A_40060472_C / T,Chr6A_40074137_T / C,Chr6A_40166723_C / A,Chr6A_40167303_C / T,Chr6A_40215215_T / A,Chr6A_40215953_C / T,Chr6A_40221161_T / C,Chr6A_40221488_A / G,Chr6A_40337284_T / A,Chr6A_40337298_A / C,Chr6A_40337421_T / A,Chr6A_40337423_T / G,Chr6A_40338076_A / C,Chr6A_40338758_A / C,Chr6A_40506300_C / T,C hr6A_40506321_C / T,Chr6A_40506324_C / T,Chr6A_40506331_T / A,Chr6A_40725196_G / T,Chr6A_40726294_T / C,Chr6A_40911777_C / G,Chr6A_40911824_G / A,Chr6A_42094322_G / T,Chr6A_43213214_C / T,Chr6A_43373971_A / G,Chr6A_43492593_C / G,Chr6A_43893593_A / C,Chr6A_43893602_G / T,Chr6A_43894056_C / G,Chr6A_44000276_G / C,Chr6A_44048146_G / T,Chr6A_44048319_A / G,Chr6A_44048435_C / T,Chr6A_44053333_T / A,Chr6A_44053340_T / A,Chr6A_44090239_G / A,Chr6A_44097189_G / C,Chr6A_44098421_T / G,Chr6A_44098537_C / T,Chr6A_44099154_A / G,Chr6A_44099737_C / T,Chr6A_44100123_G / A,Chr6A_44100370_G / A,Chr6A_44133060_T / G,Chr6A_44190237_G / C,Chr6A_44194547_A / C,Chr6A_44195303_G / A,Chr6A_44195335_C / T,Chr6A_44202592_C / T,Chr6A_44202751_C / T,Chr6A_44202787_C / T,Chr6A_44202871_G / T,Chr6A_44204089_G / C,Chr6A_44251131_C / T,Chr6A_44251133_T / C,Chr6A_44267217_A / C,Chr6A_44269212_G / A,Chr6A_44388678_A / T,Chr6A_44388716_G / A,Chr6A_44388798_C / A,Chr6A_44388947_T / C,Chr6A_44388948_G / A,Chr6A_44396031_C / T,Chr6A_44498737_C / A,Chr6A_44498768_C / T,Chr6A_44690987_G / A,Chr6A_44959511_C / A,Chr6A_44965469_A / G,Chr6A_44965589_C / A,Chr6A_44970425_A / G,Chr6A_44970710_C / A,Chr6A_44970724_A / G,Chr6A_46455487_A / T,Chr6A_46671799_T / A,Chr6A_46757983_A / G,Chr6A_47417840_C / T,Chr6A_47417984_A / T,C hr6A_47941589_T / C,Chr6A_47941595_A / C,Chr6A_48607305_C / G,Chr6A_49823106_G / A,Chr6A_49823128_A / C,Chr6A_49823137_T / C,Chr6A_49823295_G / T,Chr6A_49823324_T / C,Chr6A_50155124_G / A,Chr6A_57924628_G / A,Chr6A_57924633_C / T,Chr6A_58719398_C / T,Chr6A_60801465_G / A,Chr6A_63353751_C / G,Chr6A_63468879_T / C,Chr6A_65282416_G / A,Chr6A_66539922_G / A,Chr6A_66719724_T / C,Chr6B_3898833_A / G,C hr6B_15352022_C / G,Chr6B_15352042_A / G,Chr6B_22185325_G / A,Chr6B_23680494_C / T,Chr6B_32831732_A / G,Chr6B_32842253_A / T,Chr6B_32888329_C / G,Chr6B_32888354_G / A,Chr6B_32888374_A / G,Chr6B_32990988_C / T,Chr6B_33671195_T / G,Chr6B_34786688_T / C,Chr6B_35293164_A / G,Chr6B_36160679_A / G,Chr6B_36164377_T / A,Chr6B_36164381_C / T,Chr6B_36164894_C / T,Chr6B_36164909_A / C,Chr6B_36165013_C / T,Chr6B_36165052_A / G,Chr6B_36268264_G / A,Chr6B_36268269_C / T,Chr6B_36495744_G / C,Chr6B_36496030_A / G,Chr6B_36496297_A / G,Chr6B_36497260_T / C,Chr6B_36497374_G / A,Chr6B_36499036_T / C,Chr6B_36499605_T / A,Chr6B_36499964_C / T,Chr6B_36500453_T / G,Chr6B_36500625_G / C,Chr6B_36502150_T / C,Chr6B_36503103_C / T,Chr6B_36514327_G / T,Chr6B_36517173_G / C,Chr6B_36517213_C / A,Chr6B_36519169_A / C,Chr6B_36527090_G / A,Chr6B_36527213_T / C,Chr6B_36887956_A / G,Chr6B_36887968_A / T,Chr6B_36888104_T / G,C hr6B_36902394_T / C,Chr6B_36917736_G / A,Chr6B_36918138_A / G,Chr6B_36918141_T / C,Chr6B_36918176_C / G,Chr6B_37048141_C / T,Chr6B_37048247_A / T,Chr6B_37070274_A / T,Chr6B_37308951_T / C,Chr6B_37308981_G / A,Chr6B_37308985_A / G,Chr6B_37339356_T / G,Chr6B_37372369_C / T,Chr6B_37379078_C / T,Chr6B_37379090_T / G,Chr6B_37379111_C / G,Chr6B_37430353_A / G,Chr6B_37434000_G / C,Chr6B_37434023_G / A,Chr6B_37434045_T / A,Chr6B_37436886_G / A,Chr6B_37486683_G / T,Chr6B_37486740_C / T,Chr6B_37487509_T / C,Chr6B_37649044_A / G,Chr6B_38161051_C / T,Chr6B_38371350_G / A,Chr6B_38371367_A / G,Chr6B_38402191_T / C,Chr6B_38402199_A / G,Chr6B_38402205_G / A,Chr6B_38857636_G / A,Chr6B_39722749_A / G,Chr6B_39729980_C / A,Chr6B_39807225_A / C,Chr6B_39807782_G / C,Chr6B_39807971_A / T,Chr6B_39810551_A / G,Chr6B_39810801_A / G,Chr6B_39821282_C / G,Chr6B_39821444_A / G,Chr6B_39844248_T / C,Chr6B_39844297_G / A,Chr6B_39844326_T / G,Chr6B_39859255_T / C,Chr6B_40062037_A / G,Chr6B_40109991_A / T,Chr6B_40844487_A / C,Chr6B_40862353_G / A,Chr6B_40902423_A / G,Chr6B_41640626_T / C,Chr6B_43479913_G / A,Chr6B_43487324_G / A,Chr6B_43509495_C / T,Chr6B_43509528_G / C,Chr6B_43574176_A / C,Chr6B_43619176_C / T,Chr6B_43622245_C / G,Chr6B_43623185_G / A,Chr6B_43627897_T / C,Chr6B_43627924_C / T,Chr6B_43636249_C / T,Chr6B_43665510_G / T,Chr6B_43665584_C / A,Chr6B_43713054_A / T,Chr6B_43713067_G / A,Chr6B_43713620_G / T,Chr6B_43713648_C / T,Chr6B_43713694_C / T,Chr6B_43713708_G / A,Chr6B_43714625_G / A,Chr6B_43719407_G / A,Chr6B_43729067_C / T,Chr6B_43731751_T / A,Chr6B_43886055_A / G,Chr6B_44091642_A / G,Chr6B_44091647_A / T,Chr6B_44171700_T / G,Chr6B_44171768_G / T,Chr6B_44171951_T / C,Chr6B_44172303_C / T,Chr6B_44172655_T / C,Chr6B_44172763_C / G,Chr6B_44638427_G / A,Chr6B_57433410_C / A,Chr6B_57433412_C / A,Chr6B_59952704_T / C,Chr7A_334656_C / T,Chr7A_334837_G / A,Chr7A_2137354_G / A,Chr7A_6606273_T / A,Chr7A_7258968_G / T,Chr7A_7777288_A / G,Chr7A_7777303_G / A,Chr7A_8673731_A / T,Chr7A_8688575_C / A,Chr7A_8800843_T / A,Chr7A_9085892_G / A,Chr7A_9359644_G / C,Chr7A_9485870_G / T,Chr7A_9503163_C / T,Chr7A_9503191_T / C,Chr7A_9512579_T / C,Chr7A_9512582_G / T,Chr7A_9512640_T / C,Chr7A_9515877_C / T,Chr7A_9515964_C / A,Chr7A_9515970_G / A,Chr7A_9515980_T / C,Chr7A_9579221_C / A,Chr7A_9607728_G / A,Chr7A_9688086_A / T,Chr7A_9688165_C / T,Chr7A_9688254_T / C,Chr7A_9758968_C / T,Chr7A_9759081_C / T,Chr7A_9759109_C / T,Chr7A_9759110_T / C,Chr7A_9759135_C / T,Chr7A_9759149_A / C,Chr7A_9759158_C / G,Chr7A_9759193_T / C,Chr7A_9759397_G / A,Chr7A_9759568_C / G,Chr7A_9760086_T / C,Chr7A_9760899_T / G,Chr7A_9817524_T / A,Chr7A_9817626_A / G,Chr7A_9817655_A / G,Chr7A_9817663_C / T,Chr7A_9828442_A / G,Chr7A_9828473_G / C,Chr7A_9830955_T / C,Chr7A_10337320_C / T,Chr7A_10337327_A / G,Chr7A_10337339_C / T,Chr7A_10452558_T / C,Chr7A_10453260_A / G,Chr7A_10663636_C / T,Chr7A_10663637_A / G,Chr7A_10663672_A / G,Chr7A_10663677_G / A,Chr 7A_10663693_G / A,Chr7A_10726919_T / C,Chr7A_11112822_C / T,Chr7A_11241668_T / G,Chr7A_11281772_C / T,Chr7A_11455372_C / A,Chr7A_11513450_A / G,Chr7A_11950658_C / T,Chr7A_12239864_C / T,Chr7A_12239870_A / G,Chr7A_12239877_G / T,Chr7A_12329493_A / G,Chr7A_12336580_T / C,Ch r7A_12384508_G / A,Chr7A_12409353_A / C,Chr7A_12409357_G / T,Chr7A_12586722_A / T,Chr7A_12591039_G / A,Chr7A_12591280_C / T,Chr7A_12596057_C / T,Chr7A_12597134_G / A,Chr7A_12605091_T / G,Chr7A_12626884_T / C,Chr7A_12721192_G / A,Chr7A_12721200_C / T,Chr7A_12721202_G / A,C hr7A_12844677_T / A,Chr7A_12873509_C / T,Chr7A_12958699_C / A,Chr7A_12958702_T / A,Chr7A_12958735_G / A,Chr7A_13036224_A / G,Chr7A_13076607_G / A,Chr7A_13197998_C / T,Chr7A_13397801_A / C,Chr7A_13397805_G / A,Chr7A_13397819_A / C,Chr7A_13611136_G / A,Chr7A_13630520_G / A,Chr7A_13630667_G / C,Chr7A_13630712_C / T,Chr7A_13885707_C / T,Chr7A_13886329_G / A,Chr7A_13886380_G / A,Chr7A_13886538_A / G,Chr7A_13886626_T / C,Chr7A_13886631_A / G,Chr7A_14110745_A / C,Chr7A_14110808_A / G,Chr7A_14110828_A / G,Chr7A_14110839_T / G,Chr7A_14173676_T / C, Chr7A_14578340_T / C,Chr7A_14929504_G / C,Chr7A_15067192_G / A,Chr7A_15461255_T / A,Chr7A_15532685_A / G,Chr7A_15532700_G / A,Chr7A_15535350_A / G,Chr7A_16019923_A / G,Chr7A_16019930_T / C,Chr7A_16228109_T / C,Chr7A_16228128_C / T,Chr7A_16532016_G / T,Chr7A_16532018_C / A, Chr7A_16993391_T / C,Chr7A_17111088_T / C,Chr7A_17123196_A / C,Chr7A_17179195_T / C,Chr7A_18076755_C / T,Chr7A_18128374_G / A,Chr7A_18258710_G / A,Chr7A_18351281_A / G,Chr7A_18730874_T / A,Chr7A_18849598_C / G,Chr7A_18849603_C / T,Chr7A_19174082_C / T,Chr7A_19177811_T / C, Chr7A_19177844_T / C,Chr7A_19510925_A / G,Chr7A_19537148_C / G,Chr7A_19537153_C / T,Chr7A_19754642_T / A,Chr7A_19915141_G / T,Chr7A_20044044_G / A,Chr7A_20102329_C / G,Chr7A_20947148_T / C,Chr7A_22685510_T / C,Chr7A_26941037_A / C,Chr7A_29692370_G / A,Chr7A_30199750_T / A,Chr7A_30602738_G / A,Chr7A_30705882_C / G,Chr7A_30705914_T / C,Chr7A_30812924_C / G,Chr7A_30900558_C / T,Chr7A_30900573_C / T,Chr7A_31280296_C / G,Chr7A_31280298_T / G,Chr7A_31445522_T / C,Chr7A_31853848_C / T,Chr7A_32798105_A / G,Chr7A_32798115_C / T,Chr7A_34249664_A / C, Chr7A_34515249_G / A,Chr7A_34515262_C / A,Chr7A_35568412_C / T,Chr7A_35889563_T / C,Chr7A_37276436_G / A,Chr7A_37519939_T / C,Chr7A_37522232_C / T,Chr7A_37522770_G / A,Chr7A_37524171_G / A,Chr7A_37524700_G / A,Chr7A_37524727_C / T,Chr7A_37524736_C / T,Chr7A_37525479_G / A, Chr7A_37527825_G / T,Chr7A_37530699_G / T,Chr7A_38095606_C / T,Chr7A_38206144_T / C,Chr7A_38264719_T / C,Chr7A_38264984_G / A,Chr7A_38293193_C / T,Chr7A_38293200_C / A,Chr7A_38498105_C / T,Chr7A_38499046_C / T,Chr7A_38500173_C / T,Chr7A_38636156_G / T,Chr7A_38738734_A / G, Chr7A_38900371_T / C,Chr7A_38901629_T / C,Chr7A_38961982_C / G,Chr7A_38993890_C / T,Chr7A_39180492_C / T,Chr7A_39180494_A / G,Chr7A_39194802_G / A,Chr7A_39261125_G / T,Chr7A_43089920_G / A,Chr7A_43089958_G / A,Chr7A_57526458_T / G,Chr7A_69716363_G / A,Chr7A_69716364_G / A,Chr7B_4713402_C / T,Chr7B_8326234_G / A,Chr7B_8512948_G / A,Chr7B_8928827_A / C,Chr7B_9129457_C / T,Chr7B_9129470_G / C,Chr7B_9129475_A / G,Chr7B_9129477_G / T,Chr7B_9129490_T / C,C hr7B_9296368_G / A,Chr7B_9297304_T / A,Chr7B_9301825_C / T,Chr7B_9427761_G / A,Chr7B_9427783_C / T,Ch r7B_9428118_T / C,Chr7B_9428179_T / A,Chr7B_9428530_A / G,Chr7B_9428540_A / T,Chr7B_9428548_A / G,Chr7B_9429659_T / C,Chr7B_9429692_A / G,Chr7B_9498049_A / G,Chr7B_11052046_G / A,Chr7B_11465967_T / C,C hr7B_11465983_C / T,Chr7B_11466008_C / T,Chr7B_11466220_G / C,Chr7B_11466245_A / G,Chr7B_11466295_T / A,Chr7B_11466329_A / G,Chr7B_11466390_T / G,Chr7B_11781726_C / A,Chr7B_12057741_C / G,Chr7B_12057743_A / G,Chr7B_12057760_A / G,Chr7B_12057770_A / G,Chr7B_12057779_C / T,Chr7B_12057837_C / T,Chr7B_12057862_C / T,Chr7B_12057866_T / C,Chr7B_12057877_G / A,Chr7B_12602363_T / C,Chr7B_12602370_T / C,Chr7B_12602421_T / C,Chr7B_12602447_A / C,Chr7B_12602455_C / T,Chr7B_12730014_A / G,Chr7B_12898225_G / C,C hr7B_13180549_C / T,Chr7B_13180812_C / G,Chr7B_13180914_C / A,Chr7B_13180920_C / G,Chr7B_13237247_C / G,Chr7B_13243133_A / G,Chr7B_13243284_C / T,Chr7B_13243346_C / T,Chr7B_13243390_T / C,Chr7B_13243400 _G / C,Chr7B_13322417_T / C,Chr7B_13644163_C / T,Chr7B_13785053_G / T,Chr7B_13785064_T / C,Chr7B_1468 1449_T / C,Chr7B_14690631_A / G,Chr7B_14691044_T / A,Chr7B_15582327_T / A,Chr7B_15641713_A / G,Chr7B_ 15642078_G / A,Chr7B_15643592_T / A,Chr7B_15644255_A / G,Chr7B_15644376_T / C,Chr7B_15685901_C / G,Ch r7B_15705069_T / G,Chr7B_16286745_C / T,Chr7B_16286753_T / C,Chr7B_16650848_A / G,Chr7B_17078415_C / A,Chr7B_17078437_ G / A,Chr7B_17078439_T / A,Chr7B_17079540_C / A,Chr7B_17079541_G / T,Chr7B_17079561_A / T,Chr7B_17080016_G / C,Chr7B_1757 7258_A / G,Chr7B_17577384_T / C,Chr7B_17577385_A / G,Chr7B_17577390_C / A,Chr7B_17577403_T / C,Chr7B_17595399_G / T,Chr7B _17649546_T / C,Chr7B_24789008_C / T,Chr7B_35288831_G / A,Chr7B_35288837_G / A,Chr7B_50691298_A / T,Chr7B_52207249_C / T,

[0080] The SNP sites were determined through genome-wide association analysis of 358 rose resequencing materials and association screening using multiple software and models.

[0081] Example 2

[0082] The present invention discloses a probe for predicting wavy outer edges of rose petals. The screening principles of the probe include: being designed from a combination of SNP sites, being able to specifically bind to the SNP sites, being used to capture a target DNA region, and realizing the detection of a specific SNP site. The sequence information of the probe is associated with the SNP site combination, and conforming to various screening principles of probe design, including the absence of Indel markers within 50 bp upstream and downstream of the SNP site, having only one copy number in the entire genome of each 100 bp upstream and downstream sequence, having a GC content of 40-60%, having no short segment repeats in the sequence, and having no N bases.

[0083] Example 3

[0084] The present invention provides a prediction chip for the wavy outer edge of rose petals. The prediction chip includes probes for detecting the 1453 SNP sites. By detecting the genotype of the SNP sites in the genomic DNA of the rose sample to be tested and utilizing a pre-constructed whole-genome selection model, accurate prediction of the wavy outer edge trait of the rose petals is achieved.

[0085] Example 4

[0086] The invention discloses an application of a SNP molecular marker combination, a probe or a chip in predicting the outer edge wave of rose petals, which is achieved by extracting genomic DNA from rose plants and using the SNP site combination, the probe or the chip for detection.

[0087] Example 5

[0088] A method for predicting rose petal outer edge waves based on SNP molecular marker combinations of the present invention comprises the following steps:

[0089] (1) Extracting genomic DNA from the rose plants to be tested;

[0090] (2) constructing a library of genomic DNA;

[0091] (3) Use probes to hybridize and capture DNA fragments in the library to enrich DNA fragments containing target SNP sites; the hybridization reaction system includes the following components: 10-20 μL of Pre-PCR amplification library; 20-30 μL of liquid probe mixture, wherein the concentration of each liquid probe in the liquid probe mixture is 50-100 nM; 5-10 μL of hybridization buffer, wherein the hybridization buffer contains 3-5 mol / L salt solution and 1%-2% blocking agent; RNase-free water is added to make up the reaction system to 50-100 μL.

[0092] (4) performing high-throughput sequencing on the captured DNA fragments;

[0093] (5) Compare the sequencing data with the rose reference genome to determine the genotype of the SNP site;

[0094] (6) The genotype data of the SNP locus combination are input into the whole genome selection model to obtain the prediction results of the wavy traits of the outer edge of the petals of the tested rose.

[0095] Example 6

[0096] 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 petal outer edge wavy traits of breeding materials during the rose breeding process, thereby shortening the breeding cycle, improving selection efficiency, and cultivating new rose varieties with excellent petal outer edge wavy traits.

[0097] The whole-genome selection model constructed based on the combination of SNP sites can predict the wavy outer edge of rose petals with a prediction accuracy greater than 0.563.

[0098] Implementation 7

[0099] The present invention provides a rose molecular breeding method based on SNP markers, comprising the following steps:

[0100] (1) Collect rose samples and extract genomic DNA: Leaf samples of multiple rose varieties were collected, and total DNA was extracted using the magnetic bead method. After fragmentation and end repair, sequencing adapters were connected to construct a pre-PCR amplification library;

[0101] (2) SNP marker detection: A liquid-phase breeding chip containing specific probes is used to hybridize and capture the DNA in the sample library, specifically enrich the target SNP region DNA, and obtain the captured library after elution and purification; the captured library is subjected to high-throughput sequencing on the BGI DNBSEQ-T7 platform, and after quality control, it is aligned to the rose reference genome to determine the sample genotype data. The SNP markers are 1453 SNP markers across 14 chromosomes that are significantly associated with the petal outer edge wave, which are determined by integrating 358 rose whole genome resequencing data and systematic petal outer edge wave phenotype association analysis using a multi-model joint screening strategy;

[0102] (3) Phenotypic data collection: The phenotypic data of the outer edge of rose petals were collected, and three repeated observations were recorded according to the standardized scoring system. After calculating the BLUP value, it was combined with five types of phenotypic indicators for subsequent association analysis;

[0103] (4) Trait prediction based on whole genome selection model: Based on the sample genotype data and phenotypic data, the whole genome selection model was trained using algorithms such as LightGBM, random forest, and Bayesian ridge regression. The model was used to predict the wavy traits of the petal outer edge of the breeding material and select candidate individuals with excellent wavy traits. Among them, the Bayesian ridge regression prediction accuracy was the best (R 2 >0.563);

[0104] (5) Breeding decision-making: Based on the prediction results, select candidate individuals with excellent petal outer edge wavy traits for reproduction and breeding to accelerate the rose molecular breeding process.

[0105] Example 8

[0106] Analyze the whole-genome association analysis of petal outer edge waves of 358 rose whole-genome resequencing data, including the following process:

[0107] (1) 358 rose leaf samples were collected, and genomic DNA was purified by magnetic bead adsorption method. DNA fragments that met the length standard were screened, and after fluorescence quantification, whole genome resequencing was completed using the DNBSEQ-T7 platform.

[0108] (2) The original sequencing data (fq.gz format) were aligned to the modern rose 'Samantha' genome v2 version using BWA software

[0109] (https: / / figshare.com / articles / dataset / _i_R_hybrida_i_Samantha_genome_v2_ / 28738781?file=53454104), using the GATK pipeline for variation detection, initially obtained approximately 115 million SNP sites.

[0110] (3) Filter low-quality sites: retain allele frequency (MAF) > 0.01 ( Figure 1 ), missing rate <0.2( Figure 2 ) SNPs, ultimately yielding approximately 35.33 million high-quality loci. Data on the petal outer edge wave phenotype were collected, and three replicates were recorded using a standardized scoring system (0 represents absence, 1 represents presence). BLUP values ​​were calculated and then combined with five phenotypic indicators for GWAS analysis.

[0111] (5) Six models including EMMAX, GEMMA, and TASSEL (covering FARMCPU, GLM, and MLM algorithms) were used, and the significance threshold (p < 1 × 10 -4 ), 269,000 associated SNPs were screened, and 135,000 candidate sites were retained after deduplication.

[0112] (6) Probe feasibility assessment of candidate SNPs: exclude sites with InDels, repeat sequences, or N bases within 50 bp upstream and downstream, screen SNPs with GC content of 40%-60% and single copy regions, and finally retain 5710 highly specific sites.

[0113] (7) 1453 core SNPs across 14 chromosomes were selected based on the multi-model association strength for probe synthesis and model construction ( Figure 5 ).

[0114] (8) Based on 13 algorithms including LightGBM, random forest, and Bayesian regression, the model was trained using genotype and phenotype data of 358 samples. The results showed that Bayesian Ridge Regression (BRR) had the best prediction accuracy (R 2 >0.563), significantly better than traditional linear models (such as Ridge regression) ( Figure 4 )

[0115] (9) The SNP sites screened in step (7) are synthesized into probe sequences for capture sequencing.

[0116] Example 9

[0117] Method for detecting rose using the liquid phase probe corresponding to the 1453 SNPs described in Example 8

[0118] Genomic DNA Extraction: Fresh rose leaves were collected, rapidly frozen, and transported to the laboratory for total DNA extraction using magnetic beads. Genomic DNA Library Construction: Genomic DNA was fragmented (200-300 bp), end-repaired, and ligated with sequencing adapters to construct a pre-PCR amplification library. Probe Hybridization and Purification: Streptavidin-modified SNP probes were hybridized to the library to specifically enrich the target region DNA. After washing out non-bound fragments, magnetic bead purification was performed to obtain a high-concentration capture library.

[0119] Hybridization conditions: The streptavidin-labeled probe was hybridized with the target library at 50°C for 16 hours. The biotin-labeled magnetic beads were combined with the streptavidin-labeled probe at room temperature for 30 minutes to enrich the target library. The non-target region library was rinsed with wash buffer and eluted with nuclease-free water.

[0120] High-throughput sequencing: High-throughput sequencing was performed on the BGI DNBSEQ-T7 platform. Raw data were quality-controlled (removing low-quality reads and adapter contamination) and then aligned to the reference genome. Liquid-phase probe capture efficiency evaluation: Liquid-phase probe capture efficiency evaluation: Cleaned sequencing data were aligned to the modern rose 'Samantha' genome v2 reference sequence using bwa software to assess capture efficiency.

[0121] Test Example 1

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

[0123] Experimental Materials

[0124] Twelve modern rose varieties with significant genetic differences were selected (see Table 2 for specific numbers). Fresh leaves were immediately frozen in liquid nitrogen and stored at -80°C. Genomic DNA from each variety was extracted using the CTAB method (cetyltrimethylammonium bromide extraction technique). DNA purity (OD260 / 280 between 1.8 and 2.0) and concentration (≥50 ng / μL) were verified by agarose gel electrophoresis coupled with a Nanodrop spectrometer.

[0125] Table 2

[0126]

[0127]

[0128] Variety Representativeness: The selected materials cover six major color series (red, yellow, white, pink, purple, and orange), as well as major commercial rose types such as hybrid tea roses and floriferous roses. Red roses include Orange Flame (Test 6), Black Tornado (Test 7), and Yuna (Test 5); yellow roses are represented by Electronic Watch (Test 9); white roses include Bianca (Test 2); and pink roses include Shakra (Test 3) and Bella Vita (Test 10). This experimental design, combining multiple color series and genetic backgrounds from multiple commercial rose types, provides scientific support for the universality validation of the probe across diverse genetic backgrounds. All sample processing and DNA extraction followed standardized procedures to ensure the reliability of the experimental data.

[0129] Capture efficiency evaluation

[0130] Based on the evaluation results of 12 monthly materials, the proportion of 1453 SNPs that can be detected ranges from 97.61% to 98.78%, with a coverage depth of 376.89% to 548.15. 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.

[0131] The detection rate statistics of capture sequencing samples are shown in Table 3:

[0132] Table 3

[0133] Sample_ID Coverage Depth Detection rate (%) Labanites 387.45 97.83 Shakra 532.1 98.55 Yuna 406.72 97.92 Black Whirlwind 485.3 98.24 electronic watches 376.89 97.61 Zinderella 548.15 98.78 Bianca 412.6 98.03 Dutch ladies 463.25 98.42 Orange flame 395.55 97.95 brilliance 510.8 98.67 Bella Vita 429.75 98.11 flashback 381.2 97.72

[0134] SNP detection data for 12 rose varieties (including Laban and Shakra) showed that the detection rate for all samples was ≥97.61% (mean 98.17% ± 0.42%), indicating that ≥1418 of the 1453 SNPs detected were effectively detected. The coverage depth ranged from 376.89X to 548.15X (mean 457.36X ± 72.28X), significantly exceeding the minimum SNP typing depth threshold (≥30X). Among them, the detection rate was highest in Zinderela (Test11) (98.78%), while that in Electronic Table (Test9) was the lowest (97.61%). The detection rate coefficient of variation (CV) for Zinderela (Test11) was only 0.43%, confirming the excellent applicability of the probe across varieties.

[0135] In terms of technical performance, this liquid-phase probe system achieves: ① Target SNP capture efficiency ≥ 97.61%, meeting the whole genome selection (GS) typing accuracy requirement (> 95%); ② An average coverage depth of 457.36X can effectively reduce the risk of allele dropout; ③ Stable performance across 12 diverse varieties (detection rate fluctuation range ≤ 1.17%), demonstrating the broad applicability of the probe design.

[0136] Conclusion: This liquid-phase probe system can be efficiently applied to SNP typing of rose germplasm resources, analysis of population genetic structure, and genome-wide selection breeding practices.

[0137] 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 rose petal outer edge wavy, characterized by: The SNP molecular marker combination includes 1453 SNP sites distributed on the 14 chromosomes of roses; the specific information of the SNP sites is represented by the structure of chromosome number_physical position_allele type, and the SNP sites contain dominant alleles that are positively and significantly associated with the waves on the outer edge of rose petals. The specific site information is shown in Table 1 of the manual.

2. The genome-wide SNP molecular marker combination for rose petal outer edge wave 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 through multiple software and model associations.

3. A probe for predicting the outer edge waves of rose petals, characterized by: The screening principles of the probe include: being designed from the SNP site combination of claim 1, being able to specifically bind to the SNP site, being used to capture the target DNA region, and realizing the detection of a specific SNP site, and its sequence information being associated with the SNP site combination, and complying with various screening principles of probe design, including no Indel markers within 50 bp upstream and downstream of the SNP site, only one copy number in the entire genome of each 100 bp upstream and downstream sequence, a GC content of 40-60%, no short segment repeat sequences in the sequence, and no N bases.

4. A chip for predicting the outer edge waves of rose petals, characterized by: The prediction chip includes probes for detecting the 1453 SNP sites. By detecting the genotype of the 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 wavy traits on the outer edge of the rose petals can be 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 the outer edge wavyness of rose petals, 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 the outer edge wave of rose petals 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) The genotype data of the SNP site combination described in claim 1 is input into the whole genome selection model described in claim 4 to obtain the prediction result of the wavy trait of the outer edge of the petals of the rose to be tested.

7. The method for predicting rose petal outer edge waves using the SNP molecular marker combination according to claim 6, wherein: In step (3), the hybridization reaction system includes the following components: 10-20 μL of Pre-PCR amplification library; 20-30 μL of liquid probe mixture, wherein the concentration of each liquid probe in the liquid probe mixture is 50-100 nM; 5-10 μL of hybridization buffer containing 3-5 mol / L salt solution and 1%-2% blocking agent; RNase-free water is added to make up the reaction system to 50-100 μL.

8. 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 wavy traits of the petals' outer edges of breeding materials during the rose breeding process, thereby shortening the breeding cycle, improving selection efficiency, and cultivating new rose varieties with excellent wavy traits of the petals' outer edges.

9. Use of the SNP locus combination, probe, chip or prediction method according to claim 8 in rose molecular breeding, characterized in that: The whole genome selection model constructed based on the SNP site combination described in claim 1 can predict the outer edge waves of rose petals with a prediction accuracy greater than 0.

563.

10. A rose molecular breeding method based on SNP markers, characterized in that The steps include: (1) Collect rose samples and extract genomic DNA: Leaf samples of multiple rose varieties were collected, and total DNA was extracted using the magnetic bead method. After fragmentation and end repair, sequencing adapters were connected to construct a pre-PCR amplification library; (2) SNP marker detection: Using a liquid-phase breeding chip containing specific probes, the DNA in the sample library is hybridized and captured, the target SNP region DNA is specifically enriched, and the captured library is obtained after elution and purification; The captured library was subjected to high-throughput sequencing on the BGI DNBSEQ-T7 platform and aligned to the rose reference genome after quality control to determine the sample genotype data. The SNP markers were 1,453 SNP markers across 14 chromosomes that were significantly associated with petal outer edge wavy patterns, identified by integrating 358 rose whole-genome resequencing data with systematic petal outer edge wavy phenotype association analysis using a multi-model joint screening strategy. (3) Phenotypic data collection: The phenotypic data of the outer edge of rose petals were collected, and three repeated observations were recorded according to the standardized scoring system. After calculating the BLUP value, it was combined with five types of phenotypic indicators for subsequent association analysis; (4) Trait prediction based on whole-genome selection model: Based on the sample genotype data and phenotypic data, the whole-genome selection model was trained using algorithms such as LightGBM, random forest, and Bayesian ridge regression. The model was used to predict the wavy traits of the petal outer edge of the breeding material and select candidate individuals with excellent wavy traits. Among them, the Bayesian ridge regression prediction accuracy was the best. (5) Breeding decision-making: Based on the prediction results, select candidate individuals with excellent petal outer edge wavy traits for reproduction and breeding to accelerate the rose molecular breeding process.

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