A SNP molecular marker combination based on rosa chinensis germplasm resource typing, and an identification method and application thereof

Abstract

This invention discloses a SNP molecular marker combination based on rose germplasm resource typing, its identification method, and its application. By analyzing resequencing data from 126 Rosa genus germplasm resources, 5138 key loci were screened. Combined with phylogenetic analysis, population structure analysis, and principal component analysis, this provides important evidence for identifying the genetic composition of rose germplasm resources. This invention can accurately predict the genetic background of rose germplasm resources, providing a scientific basis for breeding work, thereby improving breeding efficiency and accuracy, shortening the breeding cycle, and reducing costs. Simultaneously, it provides a new direction for distinguishing the genetic composition of rose germplasm resources and for breeding, and has reference value for identifying the genetic composition of traits in other horticultural crops. Furthermore, the designed liquid-phase breeding chip has a highly efficient and stable capture efficiency and can be widely used for the detection and evaluation of large batches of samples.

Description

Technical Field

[0001] This invention relates to the field of molecular breeding technology, and in particular to a combination of SNP molecular markers based on rose germplasm resource typing, its identification method, and its application. Background Technology

[0002] Roses, as important ornamental plants, boast numerous varieties with complex genetic backgrounds. Traditional methods for variety identification and germplasm resource genetic background analysis primarily rely on morphological characteristics. However, this approach is susceptible to environmental factors and requires plants to reach a certain growth stage before identification can be performed, resulting in time-consuming and inefficient methods. With the development of molecular biology techniques, DNA molecular markers have become effective tools for plant variety identification and germplasm resource genetic background analysis. Single nucleotide polymorphisms (SNPs), as third-generation molecular markers, offer advantages such as abundant quantity, wide distribution, high genetic stability, and ease of automated detection.

[0003] SNPs are polymorphic sites in the genome caused by variations in a single nucleotide, which can take the form of substitution, insertion, or deletion. In plant genomics research, SNP markers are widely used in gene mapping, genetic mapping, marker-assisted selection, and genome-wide association studies. For example, in research on major crops such as maize and rice, numerous SNP markers have been developed and applied to gene mapping and molecular breeding practices, significantly improving breeding efficiency and accuracy.

[0004] Compared to traditional molecular markers such as restriction fragment length polymorphisms (RFLPs) and simple sequence repeats (SSRs), SNP markers have higher density and better genome coverage. Due to their high-density distribution throughout the genome, SNP markers can more accurately locate gene loci associated with target traits. Furthermore, SNP marker detection can be achieved through high-throughput genotyping technologies, such as microarray technology and sequencing technology. These technologies not only improve detection efficiency but also reduce detection costs. For example, SNP microarrays can simultaneously detect tens of thousands of SNP loci in a short time, providing strong technical support for large-scale genetic analysis and breeding practices.

[0005] Although SNP markers have been widely used in many plants, their application in the typing and identification of rose germplasm resources is still in its early stages. The genetic backgrounds of rose varieties are complex, and the genetic backgrounds of germplasm resources among varieties are often ambiguous. Traditional morphological identification methods are easily affected by environmental factors and require plants to reach specific growth stages for accurate identification, making them time-consuming and inefficient. Therefore, introducing SNP marker technology into the typing and identification of rose germplasm resources is of great significance and has broad application prospects.

[0006] High-throughput SNP genotyping technology allows researchers to quickly and accurately identify specific SNP loci associated with the genetic background of rose varieties, enabling precise identification and genetic analysis of germplasm resources. This marker-based identification method significantly improves variety identification efficiency, shortens the identification cycle, and reduces identification costs. Furthermore, combining this method with advanced bioinformatics techniques such as phylogenetic analysis can further clarify the genetic components and genetic distances between varieties, providing technical support for rose variety identification, resource conservation, and new variety breeding.

[0007] Currently, whole-genome resequencing is widely used for genetic background analysis of rose germplasm resources. While highly accurate, this method is expensive and complex to analyze, making it difficult to apply on a large scale in breeding practice. Existing technologies lack a method for differentiating the genetic background of rose germplasm resources that balances accuracy, economy, and ease of operation. Therefore, developing an efficient and accurate SNP molecular marker combination based on rose germplasm resource typing, along with its identification method and applications, for rose germplasm resource typing and genetic background analysis, is of great significance for rose breeding and variety conservation. Summary of the Invention

[0008] The purpose of this invention is to provide a combination of SNP molecular markers based on rose germplasm typology for distinguishing the genetic background of rose germplasm resources, as well as its identification method and application, to solve the problems of high cost and complex operation in the existing technology of genetic background analysis of rose germplasm resources.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] Firstly, this application provides an SNP molecular marker combination based on rose germplasm resource typing.

[0011] Secondly, this application provides a typing identification probe for rose germplasm resources.

[0012] Thirdly, this application provides a chip for identifying the genotype of rose germplasm resources.

[0013] Fourthly, this application provides an application of SNP molecular marker combinations, probes, or chips in predicting the genetic background of rose germplasm resources.

[0014] Fifthly, this application provides a method for identifying rose germplasm resources based on the SNP molecular marker combination.

[0015] Sixthly, this application provides an application of SNP site combinations, identification probes, identification chips, or prediction methods in the molecular breeding of roses.

[0016] Seventhly, this application provides a molecular breeding method for roses based on SNP markers.

[0017] The first aspect of this application provides a combination of SNP molecular markers for the typing and identification of rose germplasm resources. The combination of SNP molecular markers includes 5138 specific SNP loci distributed in the rose genome. The SNP loci contain specific alleles that can be used to reveal the phylogenetic relationships and population genetic structure among rose varieties. The specific information of the SNP loci is represented in the structure of chromosome number_physical location_allele type, and the specific locus information is as shown in the specification. The SNP loci are obtained by analyzing and screening whole genome resequencing data. The population genetic analysis results using these 5138 SNP loci are highly consistent with the analysis results based on all SNP loci in the whole genome.

[0018] Furthermore, the SNP sites were identified through population genetic analysis of 126 rose resequencing materials, including phylogenetic tree construction, population structure analysis, and principal component analysis.

[0019] The second aspect of this application provides an identification probe for genotyping rose germplasm resources. The identification probe is designed from a combination of SNP sites and can specifically bind to the SNP sites to capture target DNA regions, thereby enabling the detection of specific SNP sites. The probe sequence corresponds one-to-one with the SNP site combination and meets the following screening principles: the 100bp upstream and downstream sequences of the SNP site are unique in the genome and there are no Indel variations; the GC content is 40-60%; there are no short repetitive sequences and no N bases in the sequence.

[0020] A third aspect of this application provides a genotyping identification chip for rose germplasm resources. The identification chip includes identification probes for detecting the 5138 SNP loci. By detecting the genotype of the SNP loci in the genomic DNA of the rose sample to be tested, population genetics identification methods are used to accurately identify the genetic background of the rose germplasm resources.

[0021] The fourth aspect of this application provides the application of SNP molecular marker combinations, identification probes, or identification chips in the typing and identification of rose germplasm resources. After extracting genomic DNA from rose plants, the SNP site combinations, probes, or chips are used for detection and analysis.

[0022] The fifth aspect of this application provides a method for identifying rose germplasm resources based on SNP molecular marker combinations, comprising the following steps:

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

[0024] (2) Construct a library from the genomic DNA;

[0025] (3) Use probes to hybridize and capture DNA fragments in the library to enrich DNA fragments containing the target SNP site;

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

[0027] (5) Compare and analyze the sequencing data with the rose reference genome to determine the genotype of the SNP locus;

[0028] (6) Population genetic analysis based on SNP locus combination genotype data, including phylogenetic tree construction, population structure analysis and principal component analysis, to analyze the kinship and genetic background of the rose varieties to be tested.

[0029] The sixth aspect of this application provides an application of SNP site combinations, identification probes, identification chips, or identification methods in rose molecular breeding, which is used to identify and analyze the genetic background of breeding materials in an early, accurate, and efficient manner during the rose breeding process, thereby guiding parent selection, improving breeding efficiency, and shortening the breeding cycle.

[0030] The seventh aspect of this application provides a molecular breeding method for roses based on SNP markers, comprising the following steps: applying SNP locus combinations, identification probes or identification chips to the breeding process of rose varieties, analyzing the genetic background of varieties by detecting genotypes at the seed or seedling stage, thereby screening out plants with ideal genetic backgrounds and accelerating the breeding of superior new rose varieties.

[0031] Beneficial effects: This invention can shorten the breeding cycle, improve breeding efficiency, reduce costs, improve identification accuracy, and has good application prospects. Through precise analysis of the genetic components of rose varieties, it can accelerate the cultivation of new rose varieties with excellent genetic background to meet market demand.

[0032] Compared with the prior art, the present invention has the following advantages:

[0033] Significant cost-effectiveness: Compared with whole-genome resequencing, using the combination of 5138 SNP loci of this invention for genetic background analysis of germplasm resources can significantly reduce detection costs and save time and resources.

[0034] High accuracy: The 5138 specific SNP loci screened in this invention can accurately reflect the genetic relationships among rose varieties. The population genetics analysis results are highly consistent with the whole genome SNP locus analysis results, ensuring the reliability of the identification results.

[0035] (3) Easy to operate: By designing specific probes and chips, the target SNP sites can be efficiently captured and detected, simplifying the operation process and reducing the technical threshold.

[0036] (4) Broad application prospects: This invention can provide technical support for rose variety identification, genetic background analysis of germplasm resources, and screening of breeding materials, and has important application value in rose breeding and variety protection.

[0037] (5) Comprehensive analysis dimensions: Multiple population genetics methods such as phylogenetic tree construction, population structure analysis and principal component analysis are used to comprehensively analyze the genetic background of rose germplasm resources from different perspectives.

[0038] (6) Advantages of liquid phase breeding chip: The SNP sites of the present invention can be used to prepare liquid phase breeding chips. These chips have efficient technical means and can more conveniently and quickly perform gene detection and analysis on a large number of rose samples, further improving the efficiency and accuracy of breeding work.

[0039] (7) Stable capture efficiency: The results of evaluating the liquid phase probe capture efficiency of 12 seasonal varieties in Example 3 showed that the proportion of 5138 SNPs that could be detected ranged from 97.62% to 98.81%, with a coverage depth of 391.08 to 531.36X. The overall capture efficiency was good, indicating that the probe and chip of the present invention have good stability and reliability. 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 The distribution map of 5138 SNP loci on the 14 chromosomes of rose provided by this invention.

[0042] Figure 2 Allelic frequency map of the genetic background of rose germplasm resources provided by this invention.

[0043] Figure 3 A graph showing the missing rate of the genetic background of rose germplasm resources provided by this invention.

[0044] Figure 4 Heterozygosity diagram of the genetic background of rose germplasm resources provided by this invention.

[0045] Figure 5 Phylogenetic tree of the genetic background of rose germplasm resources provided by this invention.

[0046] Figure 6 Principal component analysis diagram of the genetic background of rose germplasm resources provided by this invention.

[0047] Figure 7 Different K-value diagrams for the rose germplasm population structure provided by this invention.

[0048] Figure 8 A population structure analysis diagram of rose germplasm provided for this invention.

[0049] Figure 9 Principal component analysis diagram of the genetic background of the rose germplasm resources to be tested provided by this invention.

[0050] Figure 10 Phylogenetic tree of the genetic background of the rose germplasm resources to be tested provided by this invention. Detailed Implementation

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] The terms "first" and "second" are used for descriptive purposes only, to distinguish objects, such as substances, from one another, and should not be construed as indicating or implying relative importance or implicitly specifying the diameter of the indicated technical features. For example, without departing from the scope of the embodiments of this application, "first XX" may also be referred to as "second XX," and similarly, "second XX" may also be referred to as "first XX." Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of that feature.

[0058] The first aspect of this application provides a combination of SNP molecular markers based on rose germplasm resource typing. The combination of SNP molecular markers includes 5138 SNP loci distributed on the 14 chromosomes of rose. The SNP loci contain specific alleles that can be used to reveal the phylogenetic relationship and population genetic structure among rose varieties. The specific information of the SNP loci is represented in the structure of chromosome number_physical location_allele type. The specific locus information is as shown in the specification.

[0059] SNP loci were identified through population genetic diversity analysis of 126 rose resequencing materials, followed by phylogenetic tree construction, population structure analysis, and principal component analysis.

[0060] The second aspect of this application provides an identification probe for genotyping rose germplasm resources. The identification probe is designed from a combination of SNP sites and can specifically bind to the SNP sites to capture target DNA regions, thereby enabling the detection of specific SNP sites. The probe sequence corresponds one-to-one with the SNP site combination and meets the following screening principles: the 100 bp upstream and downstream sequences of the SNP site are unique in the genome and there are no Indel variations; the GC content is 40-60%; there are no short repetitive sequences and no N bases in the sequence.

[0061] The third aspect of this application provides an identification chip for genotyping rose germplasm resources. The identification chip includes probes for detecting the 5138 SNP sites. By detecting the genotype of the SNP sites in the genomic DNA of the rose sample to be tested, population genetics identification methods are used to achieve accurate analysis of the genetic background, kinship and population structure of rose varieties.

[0062] The fourth aspect of this application provides an application of SNP molecular marker combinations, probes, or chips in the genetic background analysis of rose germplasm resources, which involves extracting genomic DNA from rose plants and detecting it using the SNP site combinations, probes, or chips.

[0063] The fifth aspect of this application provides a method for identifying rose germplasm resources based on SNP molecular marker combinations, comprising the following steps:

[0064] (1) Extract genomic DNA from the rose plants to be tested;

[0065] (2) Construct a library from the genomic DNA;

[0066] (3) Use probes to hybridize and capture DNA fragments in the library to enrich DNA fragments containing the target SNP site;

[0067] (4) Perform high-throughput sequencing on the captured DNA fragments;

[0068] (5) Compare and analyze the sequencing data with the rose reference genome to determine the genotype of the SNP loci;

[0069] (6) Input the genotype data of SNP loci combination into the population genetics analysis process, and obtain the analysis results of the genetic background, kinship and population structure of the rose germplasm resources to be tested by constructing a phylogenetic tree, performing population structure analysis and principal component analysis.

[0070] In some embodiments, the phylogenetic tree and principal component analysis constructed based on the 5138 SNP sites can clearly distinguish different rose populations, and the population structure analysis results are stable and reliable.

[0071] The sixth aspect of this application provides an application of SNP locus combinations, identification probes, identification chips, or identification methods in the identification and breeding of rose germplasm resources. This is used to accurately and efficiently identify and analyze the genetic background, kinship, and population structure of breeding materials during the rose breeding process, thereby guiding the selection of parents, clarifying the genetic composition of breeding materials, and accelerating the cultivation of new rose varieties with target traits.

[0072] The seventh aspect of this application provides a method for molecular breeding of roses based on SNP markers, including the following steps: applying SNP locus combinations, identification probes or identification chips to the breeding process of rose varieties, detecting genotypes during the seed or seedling stage, analyzing the genetic background of the varieties, thereby screening out plants with ideal genetic backgrounds and accelerating the breeding of superior new rose varieties.

[0073] Example

[0074] This invention provides a SNP molecular marker combination based on rose germplasm resource typing. The SNP molecular marker combination includes 5138 SNP loci distributed on the 14 chromosomes of rose. The SNP loci contain specific alleles that can be used to reveal the phylogenetic relationships and population genetic structure among rose varieties. The specific information of the SNP loci is represented in the structure of chromosome number_physical location_allele type. The specific locus information is shown in Table 1.

[0075] Table 1

[0076] SNP loci were identified through population genetic analysis of 126 rose resequencing materials, followed by phylogenetic tree construction, population structure analysis, and principal component analysis.

[0077] Example 2

[0078] This invention discloses an identification probe for genotyping rose germplasm resources. The identification probe is designed from a combination of SNP sites and can specifically bind to the SNP sites to capture target DNA regions, thereby enabling the detection of specific SNP sites. The probe sequence corresponds one-to-one with the SNP site combination and meets the following screening principles: the 100 bp upstream and downstream sequences of the SNP site are unique in the genome and there are no Indel variations; the GC content is 40-60%; there are no short repetitive sequences and no N bases in the sequence.

[0079] Example 3

[0080] The present invention provides a genetic background identification chip for rose germplasm resources. The chip includes probes for detecting 5138 SNP loci. By detecting the genotype of SNP loci in the genomic DNA of the rose sample to be tested, population genetics identification methods are used to achieve accurate analysis of the genetic background, kinship and population structure of rose varieties.

[0081] Example 4

[0082] The present invention relates to the application of a combination of SNP molecular markers, identification probes, or identification chips in the prediction of the genetic background of rose germplasm resources. This involves extracting genomic DNA from rose plants and then using the SNP site combination, probes, or chips for detection.

[0083] Example 5

[0084] The present invention provides a method for identifying rose germplasm resources based on SNP molecular marker combinations, comprising the following steps:

[0085] (1) Extract genomic DNA from the rose plants to be tested;

[0086] (2) Construct a library from the genomic DNA;

[0087] (3) Use probes to hybridize and capture DNA fragments in the library to enrich DNA fragments containing the target SNP site;

[0088] (4) Perform high-throughput sequencing on the captured DNA fragments;

[0089] (5) Compare and analyze the sequencing data with the rose reference genome to determine the genotype of the SNP loci;

[0090] (6) The genotype data of the SNP locus combinations were input into the population genetics analysis process. By constructing a phylogenetic tree, performing population structure analysis and principal component analysis, the genetic background and population structure of the rose germplasm resources to be tested were obtained. Based on the analysis results of the 5138 SNP loci, different rose populations can be clearly distinguished, and the population genetic structure analysis is stable and reliable.

[0091] Example 6

[0092] The present invention relates to the application of an SNP locus combination, probe, chip, or identification method in the identification and breeding of rose germplasm resources. This method is used to accurately and efficiently identify and analyze the genetic background, kinship, and population structure of breeding materials during the rose breeding process, thereby guiding the selection of parent lines, clarifying the genetic composition of breeding materials, and accelerating the cultivation of target new rose varieties.

[0093] Example 7

[0094] The present invention provides a molecular breeding method for roses based on SNP markers, comprising the following steps: applying SNP locus combinations, identification probes or identification chips to the selection process of rose varieties, detecting genotypes at the seed or seedling stage, analyzing the kinship and genetic background of varieties, thereby screening out plants with ideal genetic backgrounds.

[0095] Example 8

[0096] Population genetics analysis was performed on 126 whole-genome resequencing data of rose plants, including the following workflow:

[0097] (1) Genomic DNA was extracted from 126 rose tissue samples using the magnetic bead method, and whole-genome resequencing libraries were constructed. DNA fragments that met the sequencing requirements were screened using magnetic beads. The DNA samples were quantified using a Qubit instrument and sequenced using the BGI DNBSEQ-T7 platform.

[0098] (2) The second-generation sequencing data in fq.gz format obtained in step (1) was compared with the genome of the modern rose 'Samantha' v2 version using BWA software. SNP variation data were analyzed using GATK software, ultimately identifying 135,881,593 high-quality SNP loci.

[0099] (3) The SNP sites selected in step (2) were further filtered, and those with an allele frequency greater than 0.05 and a deletion rate less than 0.1 were retained to obtain 27,813,061 high-quality SNP sites. For example Figure 2 and Figure 3 As shown, Figure 2 This is an allele frequency map of the genetic background of the rose germplasm resources provided by the present invention. Figure 3 A graph showing the missing rate of the genetic background of rose germplasm resources provided by this invention.

[0100] (4) Based on the high-quality SNP dataset obtained in step (3), population genetics analysis is performed, including phylogenetic tree construction, population structure analysis and principal component analysis, to reveal the genetic relationships and population differentiation among rose varieties;

[0101] (5) Screen for population-representative specific markers from whole-genome SNPs to design efficient analytical probes.

[0102] (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.

[0103] (7) The SNP sites selected in step (6) are optimized based on their information content and distribution uniformity in population genetics analysis, and finally 5138 representative SNP markers are determined. Figure 1 The distribution map of whole chromosome markers for the genetic background of rose germplasm resources provided by this invention.

[0104] (8) Evaluate the efficacy of the SNP loci selected in step (7) in population genetics analysis. The selected SNP loci showed good analytical ability in population genetics analysis. Phylogenetic tree ( Figure 5 The results show that, based on this SNP combination, the rose population can be clearly divided into 9 groups with a clear hierarchical structure. Principal component analysis ( Figure 6 This further validated the clustering effect of the SNP combination, showing clear separation among the nine groups in the principal component space. Furthermore, population structure analysis revealed that when the K value was 7 ( Figure 7 The genetic structures of different rose populations can be effectively distinguished. Figure 8 Furthermore, the analysis results showed high stability and reliability, indicating that the selected SNP markers are suitable for genetic analysis of rose populations.

[0105] (9) The SNP sites selected in step (7) are used to synthesize probe sequences for capture sequencing.

[0106] Example 9

[0107] The method for detecting roses using a liquid phase probe corresponding to 5138 SNPs as described in Example 8

[0108] Genomic DNA extraction: Rose leaves were collected, stored with ice packs, and transported back to the laboratory in a timely manner. Total DNA from the roses was extracted using the magnetic bead method.

[0109] Genomic DNA library construction: Genomic DNA fragmentation (200-300 bp), DNA fragment end repair and adapter ligation, Pre-PCR amplification library.

[0110] Probe hybridization with target region: The prepared SNP probe is hybridized with the Pre-PCR library. The streptomycin-modified probe is complementary to and captures the complementary DNA library. After enriching the target DNA library, other library DNAs are eluted. That is, after the captured Pre-PCR library is purified by magnetic beads, it is amplified and enriched to the DNA concentration required for high-throughput sequencing, and then high-throughput sequencing is performed.

[0111] High-throughput sequencing: The library obtained after capture and amplification in step (4) was 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 was cleaned.

[0112] Liquid phase probe capture rate assessment: The cleaned sequencing data were aligned to the reference sequence of the modern rose 'Samantha' genome v2 version using BWA software to assess its capture efficiency.

[0113] Experimental Example 1

[0114] Evaluation of liquid phase probe capture efficiency for 12 seasonal varieties

[0115] Experimental materials

[0116] Twelve rose leaf samples were selected, refrigerated, and promptly transported to the laboratory. Total DNA was extracted from each individual plant using the CTAB method. The sample numbers are shown in Table 2 below.

[0117] Table 2

[0118] (1) Assessment of capture efficiency

[0119] Based on the evaluation results of the 12-monthly rose materials, the proportion of detectable SNPs (5138) ranged from 98.45% to 99.01%, with a coverage depth of 415.58 to 523.16X, indicating good overall capture efficiency. The detection rate of captured sequencing samples is shown in Table 3. Based on the high-quality sequencing data, we further analyzed the population genetic structure and phylogenetic relationships among the 12-monthly rose varieties using principal component analysis (PCA) and phylogenetic tree construction. Principal component analysis results (e.g.) Figure 9 The diagram clearly shows the genetic composition and background differences among different varieties, while the phylogenetic tree (as shown) clearly illustrates these differences. Figure 10 The results (shown in the image) visually reveal the evolutionary relationships between varieties. These analyses provide crucial genomic evidence for understanding the genetic diversity of rose germplasm.

[0120] This experiment focused on analyzing the detection index characteristics of different samples. Twelve samples were carefully selected, including Angela, Beijing Red, and Golden Rabbit varieties. These samples are diverse and representative of the diversity within the research scope. The experiment employed professional high-throughput sequencing technology to perform depth detection on each sample, obtaining data on coverage depth and detection rate. During the detection process, standardized operating procedures were strictly followed. Multiple repeated tests were used to reduce errors, and quality control samples were used to calibrate the results, ensuring the accuracy and reliability of the data. After detection, bioinformatics analysis tools were used to process and statistically analyze the raw data, ultimately yielding the coverage depth and detection rate values ​​for each sample.

[0121] Table 3 presents the coverage depth and detection rate results for 12 samples. The table header includes three columns: "Variety Name," "Coverage Depth," and "Detection Rate (%)," clearly defining the data categories. Among them, the "Angela" sample had the highest coverage depth at 523.16, indicating the most comprehensive coverage during the testing process; the "Double Happiness" sample had the lowest coverage depth at only 415.5; "Endless Love" ranked first with 99.01%; and "Aunt Maggie" had a relatively low detection rate of 98.45%. Overall, although the coverage depth and detection rate values ​​varied among the samples, the detection rates remained at a high level. These data can provide important references for subsequent research on sample characteristics, screening of high-quality samples, or optimization of detection techniques, and help to further explore the biological or other disciplinary significance behind the samples.

[0122] Table 3

[0123] 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. Application of a SNP molecular marker combination in genetic background of Rosa chinensis germplasm resources, characterized in that: Genomic DNA was extracted from the rose plants to be tested, and SNP molecular marker combinations were used for detection and analysis. The SNP molecular marker combinatorial group comprises 5138 specific SNP loci distributed in the rose genome. This combinatorial group contains specific alleles that can be used to reveal phylogenetic relationships and population genetic structure among rose varieties. The specific information of the SNP molecular marker combinatorial group is represented using a chromosome number_physical location_allele genotype structure, as shown in Table 1. The SNP molecular marker combinatorial group was obtained through analysis and screening of whole-genome resequencing data. The population genetic analysis results using these 5138 SNP loci showed high consistency with the analysis results based on all SNP loci in the whole genome. The reference genome for the SNP molecular marker combinatorial group is the v2 version of the modern rose 'Samantha' genome.

2. The application of the SNP molecular marker combination according to claim 1 in the genetic background of rose germplasm resources, characterized in that: The SNP loci were identified through population genetic analysis of 126 rose resequencing materials, followed by phylogenetic tree construction, population structure analysis, and principal component analysis.

3. The application of the SNP molecular marker combination according to claim 1 in the genetic background of rose germplasm resources, characterized in that: The identification chip is used to perform population genetic identification, which enables accurate identification of the genetic background of rose germplasm resources. The identification chip contains probes for detecting the 5138 SNP molecular marker combinations described in claim 1, by detecting the genotype of SNP sites in the genomic DNA of the rose sample to be tested.

4. A method for identifying the genotypes of Rosa hybrida germplasm based on the SNP molecular marker combination of claim 1, characterized by Includes the following steps: (1) Extract genomic DNA from the rose plants to be tested; (2) Construct a library from the genomic DNA; (3) Using the probe with the combination of 5138 SNP molecular markers as described in claim 1, the DNA fragments in the library are hybridized and captured to enrich the DNA fragments containing the target SNP sites. (4) Perform high-throughput sequencing on the captured DNA fragments; (5) The sequencing data are compared and analyzed with the rose reference genome to determine the genotype of the SNP molecular marker combination; the rose reference genome is the reference sequence of the v2 version of the modern rose 'Samantha' genome; (6) Based on the genotype data of the SNP locus combination, perform population genetic analysis, including constructing a phylogenetic tree, population structure analysis and principal component analysis, to analyze the genetic background of the rose germplasm resources to be tested.

5. The use of a SNP molecular marker combination in the molecular breeding of Rosa hybrida, characterized by: This technology is used to identify and analyze the genetic background of breeding materials in the early, accurate, and efficient process of rose breeding, thereby guiding parental selection, improving breeding efficiency, and shortening the breeding cycle. The SNP molecular marker combination includes 5138 specific SNP loci distributed in the rose genome. The SNP molecular marker combination contains specific alleles that can be used to reveal the phylogenetic relationships and population genetic structure among rose varieties. The specific information of the SNP molecular marker combination is represented in the structure of chromosome number_physical location_allele type, and the specific locus information is shown in Table 1. The SNP molecular marker combination was obtained by analyzing and screening whole-genome resequencing data. The population genetic analysis results using these 5138 SNP loci are highly consistent with the analysis results based on all SNP loci in the whole genome. The reference genome for the SNP molecular marker combination is the v2 version of the modern rose 'Samantha' genome.

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