Chinese cherry resource core SNP (single nucleotide polymorphism) molecular marker and DNA (deoxyribonucleic acid) fingerprint database thereof

Abstract

The invention relates to the field of cherry molecular biotechnology and molecular assisted breeding technology detection, in particular to a molecular marker for identifying the core SNP (single nucleotide polymorphism) of a Chinese cherry resource and a DNA (deoxyribonucleic acid) fingerprint spectrum database constructed by using marker sites. 185 resources representing the distribution and genetic diversity of Chinese cherries in China are utilized to perform whole genome re-sequencing and biological information analysis, 469776 SNP molecular markers are obtained, 1674 SNP molecular markers are screened out to serve as core SNP markers, and a DNA fingerprint database of cherry resources is constructed and can be used for identifying germplasm resources of Chinese cherries. DNA genetic information and genetic differences of the to-be-detected Chinese cherry resources are evaluated, and a scientific basis is provided for effective protection of the Chinese cherry resources, utilization of local germplasm and selection of cherry breeding.

Description

Technical Field

[0001] The invention belongs to the field of cherry molecular biotechnology and molecular-assisted breeding technology detection, and specifically relates to core SNP molecular markers and a DNA fingerprint database that can be used to identify Chinese cherry resources. Background Art

[0002] Chinese cherry [Cerasus pseudocerasus (Lindl.) G.Don], also known as small cherry, belongs to the genus Cerasus of the Rosaceae family. It originated and was domesticated in my country. It has a cultivation history of more than 3,000 years in my country and is an important characteristic economic fruit tree in my country. Its flowers are gorgeous, its fruits are exquisite and translucent, and its flavor is rich. It has extremely high ornamental and nutritional value. It is known as the "first branch of spring fruit" and is deeply loved by consumers. At present, it has become one of the most important carriers in my country's agricultural tourism industry. Chinese cherry is widely distributed in my country and has a wide range of ecological adaptability and stress resistance. At the same time, important agronomic traits such as flower color, fruit color, sugar and acid flavor are rich in variation, and it is an important material for intra-specific and inter-specific germplasm innovation in cherries. my country's small cherry industry is mainly based on local germplasm. In production, due to the mixing of germplasm and blind introduction of species, some resources have problems such as homonyms and homonyms, and it is urgent to identify its germplasm.

[0003] The genetic information of germplasm, resource identification, and the clarification of the genetic structure and kinship of resource populations are the prerequisites for the effective protection and efficient utilization of germplasm resources, and are also an important reference for molecular breeding selection. Single nucleotide polymorphisms (SNPs), as the most abundant co-dominant marker in plant genomes, have the advantages of multiple sites, high variation, no influence from the external environment, and stable inheritance. With the development of molecular biology technology and the substantial reduction in the cost of DNA sequencing, SNPs based on the whole genome have become one of the most important molecular markers for the precise identification of germplasm resources and genetic analysis of resource populations. At present, there are no reports or applications on the development of core SNP molecular markers based on the whole genome level for Chinese cherry and the construction of a DNA fingerprint database using this marker site. Therefore, the development of core SNP molecular markers for Chinese cherry resources and the construction of a DNA fingerprint database for Chinese cherry resources are of great significance for the effective protection of cherry resources originating in my country, the efficient utilization of local germplasm, and cherry breeding selection. Summary of the invention

[0004] In response to the needs in the above-mentioned fields, the present invention provides 1674 core SNP molecular markers of Chinese cherry resources. This marker combination can truly reflect the genetic information of the Chinese cherry resource population and the genetic differences between resources. The DNA fingerprint map constructed with this marker site can effectively identify Chinese cherry resources and improve the efficiency of Chinese cherry germplasm identification. Therefore, the core SNP molecular markers of Chinese cherry resources and their DNA fingerprint map database provide a solid molecular marker and breeding selection tool for the protection, development and utilization of Chinese cherry resources, and have good practical application value.

[0005] The core SNP molecular marker site composition of Chinese cherry [Cerasus pseudocerasus (Lindl.) G.Don] resources is characterized in that the SNP molecular marker site composition is composed of 1674 SNP molecular marker sites distributed on chr1 to chr8, and the SNP site information is shown in Table 1; taking SNP1 as an example, the type G\A indicates that the reference genome of the site is G, the variable site type is A, and the rest of the sites are deduced by analogy;

[0006] The physical positions of the above-mentioned SNP molecular markers are based on the genome of the Chinese cherry Luoyang ancient cherry (Cerasus pseudocerasus'LuYg') as the reference genome and have been uploaded to the CNGB database (Accession Number: CNP0006741).

[0007] The development of the core SNP molecular markers of the Chinese cherry [Cerasus pseudocerasus (Lindl.) G.Don] resource and the construction of a DNA fingerprint database based on the marker loci include the following steps:

[0008] (1) Genomic DNA extraction: Extract the whole genome DNA of the sample to be tested;

[0009] (2) Whole genome resequencing: Whole genome resequencing is performed on the genomic DNA obtained in step (1) to obtain double-end reads;

[0010] (3) Raw reads filtering: Perform data filtering on the paired-end reads obtained in step (2) to obtain high-quality reads;

[0011] (4) SNP detection: The high-quality reads obtained in step (3) are aligned with the reference genome to perform variation detection and SNP marker screening;

[0012] (5) Discovery of high-quality SNP marker loci: Filter the SNP marker loci obtained in step (4) according to conditions such as sequencing depth, missing data, locus type, and whether they deviate from Hardy-Weinberg equilibrium to obtain the SNP marker loci after secondary screening;

[0013] (6) Evaluation of core SNP molecular marker loci and construction of DNA fingerprint maps: Further filter the SNP marker loci obtained in step (5) according to missing loci and physical positions to obtain a combination of core SNP molecular marker loci, use this to detect the population genetic structure of Chinese cherry resources, conduct principal component analysis PCA, construct a clustering tree, clarify the genetic relationship, and construct a DNA fingerprint map database.

[0014] In step (1), the improved CTAB method is used to extract genomic DNA from the sample to be tested, detect the DNA concentration and purity in a nucleic acid protein analyzer, and detect the integrity of DNA by 1% agarose gel electrophoresis;

[0015] In step (2), a cDNA library is constructed, and whole-genome resequencing is performed based on the Illumina 2000 platform to obtain raw reads;

[0016] In step (3), the raw reads obtained in step (2) are subjected to quality inspection in the Fastp software, adapters and low-quality reads are removed to obtain high-quality reads;

[0017] In step (4), the high-quality reads obtained in step (3) are aligned to the reference genome in the BWA software, sorted and marked for repetitive sequences, and then variant detection is carried out using the GATK and Sentieon software to obtain the original gvcf file, from which SNP loci are extracted for quality control, and the quality control parameters are: -filter QD<2.0 -filter FS>60.0 -filter MQ<40.0 -filter MQRankSum<-12.5 -filter ReadPosRankSum<-8.0;

[0018] The method for discovering high-quality SNP markers in step (5): In the VCFtools software, the SNPs obtained in step (4) are strictly screened according to conditions such as sequencing depth, missing data, locus type, and whether they deviate from Hardy-Weinberg equilibrium to obtain 469,776 SNP markers after secondary screening. The specific parameters are: --maf 0.01 --max-missing 0.9 --mac 3 --minDP 3 --minQ 30 --min-alleles 2 --max-alleles 2 --hwe 0.001;

[0019] Among them, the method for obtaining the core SNP molecular marker loci in step (6): (i) The SNP marker loci are evenly distributed on eight linkage groups (chr1 - chr8); (ii) There is no missing data for all SNP marker loci; (iii) The linkage disequilibrium decay (LD decay) value at the species level of Chinese cherry calculated based on 469,776 SNP marker loci is 50 bp. Therefore, there are no other SNP mutation loci within 50 bp before and after the retained marker loci, obtaining 1,674 combinations of core SNP molecular marker loci, conducting population genetic structure detection, principal component and clustering tree analysis of Chinese cherry resources, conducting comparative analysis with the relevant results of the aforementioned 469,776 SNP molecular markers, and constructing a DNA fingerprint database based on this.

[0020] Among them, in step (iii), for the 1,674 combinations of core SNP molecular marker loci screened from 469,776 SNP molecular marker loci, the methods for conducting population genetic structure detection, principal component and clustering tree analysis of Chinese cherry resources are as follows: Conduct population genetic structure detection of Chinese cherry resources in the ADMIXTURE software, perform principal component analysis PCA using the EIGENSOFT software, calculate the genetic distance between individuals in VCF2Dis and generate the final neighbor-joining method (NJ) clustering tree using phylip. Finally, sort and organize the 1,674 core SNP molecular marker loci according to the chromosome and physical positions of the loci, and construct a DNA fingerprint for each individual based on the variation type and genotype.

[0021] The test sample is young leaves.

[0022] The above SNP molecular marker locus composition is used in one or more aspects of (A) identifying Chinese cherry resources; (B) evaluating the DNA genetic information and genetic differences of Chinese cherry resources; (C) analyzing the genetic relationships, population genetic structure and genetic diversity of Chinese cherry resources; (D) constructing the core germplasm and DNA fingerprint database of Chinese cherry; (E) molecular assisted selection in Chinese cherry breeding.

[0023] The above DNA fingerprint database is used in one or more aspects of (a) identifying Chinese cherry resources; (b) evaluating the DNA genetic information and genetic differences of Chinese cherry resources; (c) analyzing the genetic relationships, population genetic structure and genetic diversity of Chinese cherry resources; (d) molecular assisted selection in Chinese cherry breeding.

[0024] The implementation purpose of the present invention is to provide a core SNP molecular marker locus composition of Chinese cherry resources and its DNA fingerprint database. It includes the following three contents:

[0025] 1. Using 185 resources representing the distribution and genetic diversity of Chinese cherry in China, whole-genome resequencing and bioinformatics analysis were carried out to obtain 469,776 SNP molecular marker loci, and 1,674 of them were selected as core SNP marker loci. The locus information is shown in Table 1.

[0026] Table 1 List of information on 1,674 core SNP molecular marker loci of Chinese cherry

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[0028]

[0029]

[0030]

[0031] 2. Provided are applications in one or more of the following aspects: (1) identifying Chinese cherry resources; (2) evaluating the DNA genetic information and genetic differences of Chinese cherry resources; (3) analyzing the genetic relationships, population genetic structures and genetic diversities of Chinese cherry resources; (4) constructing a core germplasm and DNA fingerprint database of Chinese cherry; (5) molecular-assisted selection in Chinese cherry breeding.

[0032] 3. Provided is a method for developing core SNP molecular markers of Chinese cherry resources and constructing and applying a DNA fingerprint database thereof, including the following steps:

[0033] (1) Genomic DNA extraction: The genomic DNA of the sample to be tested was extracted by the improved CTAB method, and the DNA concentration and purity were detected by a nucleic acid protein analyzer, and the integrity of the DNA was detected by 1% agarose gel electrophoresis;

[0034] (2) Whole-genome resequencing: A cDNA library was constructed from the genomic DNA obtained in step (1), and whole-genome resequencing was carried out based on the Illumina 2000 platform to obtain raw reads;

[0035] (3) Raw reads filtering: The raw reads obtained in step (2) were subjected to quality detection in the Fastp software, and the adapters and low-quality reads were removed to obtain high-quality reads. Software parameters: --qualified_quality_phred 15 --unqualified_percent_limit 40 --n_base_limit 5 --detect_adapter_for_pe;

[0036] (4) SNP detection: Align the high-quality reads obtained in step (3) to the reference genome in the BWA software, perform sorting and marking of repetitive sequences, and then use the GATK and Sentieon software to carry out variant detection to obtain the original gvcf file. Extract SNP sites from it for quality control, and the quality control parameters are: -filter QD<2.0 -filter FS>60.0 -filter MQ<40.0 -filter MQRankSum<-12.5 -filter ReadPosRankSum<-8.0;

[0037] (5) Discovery of high-quality SNP marker sites: In the VCFtools software, strictly screen the SNPs obtained in step (4) according to conditions such as sequencing depth, missing data, site type, and whether they deviate from Hardy-Weinberg equilibrium to obtain 469,776 high-quality SNP marker sites. The specific parameters are: --maf 0.01 --max-missing 0.9 --mac 3 --minDP 3 --minQ 30 --min-alleles 2 --max-alleles 2 --hwe 0.001;

[0038] (6) Evaluation of core SNP molecular marker sites and construction of DNA fingerprint maps: For the SNP molecular marker sites obtained in step (5), further screen according to the following principles: (i) The SNP marker sites are evenly distributed on eight linkage groups (chr1 - chr8); (ii) There is no missing data for all SNP marker sites; (iii) The linkage disequilibrium decay (LD decay) value at the species level of Chinese cherry calculated based on 469,776 SNP marker sites is 50 bp. Therefore, there are no other SNP mutation sites within 50 bp before and after the retained marker sites. Thus, 1,674 core SNP molecular marker site combinations of Chinese cherry resources are obtained. Based on the aforementioned 469,776 SNP molecular marker sites and the 1,674 core SNP molecular marker sites screened from them, detect the population genetic structure of Chinese cherry resources in the ADMIXTURE software, perform principal component analysis (PCA) using the EIGENSOFT software, calculate the genetic distance between individuals in VCF2Dis and generate the final neighbor-joining method (NJ) clustering tree using phylip. Finally, sort and organize the 1,674 core SNP molecular marker sites according to their chromosomal and physical positions, and construct a unique DNA fingerprint map for each individual according to the variant type and genotype.

[0039] The present invention has the following advantages and effects:

[0040] The above technical solution obtained 1,674 core SNP markers of Chinese cherry through screening, and further constructed a DNA fingerprint map of Chinese cherry using these marker loci. The population genetics results based on the aforementioned 1,674 core SNP markers are highly consistent with the results of 469,776 SNP molecular markers, indicating that this combination of core SNP molecular markers can truly reflect the comprehensive genetic information of 185 Chinese cherry resources (123 local germplasms and 62 wild resources), and has a high accuracy rate. At the same time, the DNA fingerprint map constructed based on these 1,674 core SNP molecular marker loci can effectively identify 185 Chinese cherry resources, improve the identification efficiency of Chinese cherry varieties, provide a solid molecular detection tool for the protection of Chinese cherry resources, the identification of local germplasms and breeding selection, and has good practical application value.

[0041] Attached drawings and explanations

[0042] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the accompanying drawings required for the description of the embodiments. Obviously, the accompanying drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can also be obtained based on the provided drawings.

[0043] Figure 1 Distribution of 1,674 core SNPs in the Chinese cherry genome in the embodiments of this application.

[0044] Figure 2 Population genetic structure of 185 Chinese cherry resources in the embodiments of this application.

[0045] Figure 3 Principal component analysis of 185 Chinese cherry resources in the embodiments of this application.

[0046] Figure 4 NJ clustering tree of 185 Chinese cherry resources in the embodiments of this application.

[0047] Figure 5 DNA fingerprint map of 185 Chinese cherry resources in the embodiments of this application. Specific implementation manners

[0048] To make the purpose, technical solutions and advantages of the present invention clearer, the following will further elaborate on the present invention in combination with the embodiments and the accompanying drawings. The illustrative embodiments of the present invention and their descriptions are only used to explain the present invention and are not intended to limit the present invention.

[0049] The present invention provides core SNP molecular markers for Chinese cherry resources and constructs a DNA fingerprint database using these marker sites. The information of the molecular marker sites is shown in Table 1, with specific numbers from SNP1 to SNP1674. The above SNP molecular marker site combinations have been strictly screened as core SNP markers for Chinese cherry, with high site quality and strong representativeness, which can truly reflect the genetic information and genetic differences at the whole genome level of Chinese cherry resources. They can be applied in one or more aspects such as the identification of Chinese cherry resources, the evaluation of DNA genetic information and genetic differences of Chinese cherry resources, the analysis of genetic relationships, population genetic structure and genetic diversity of Chinese cherry resources, the construction of core germplasm and DNA fingerprint databases of Chinese cherry, and molecular-assisted selection in Chinese cherry breeding.

[0050] 1. Materials and Methods

[0051] 1.1. Experimental Materials

[0052] This experiment included 123 local germplasms of Chinese cherry and 62 wild resources of Chinese cherry, a total of 185 Chinese cherry resources (Table 2). These samples covered the main natural distribution geographical regions of Chinese cherry such as the Longmenshan seismic zone, the Qinling Mountains, the Yunnan-Guizhou Plateau, and the North China and East China Plains, representing the main phenotypes and genotypes of Chinese cherry.

[0053] Table 2 Information of 185 Chinese Cherry Resources

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[0055]

[0056] 1.2 Experimental Methods

[0057] 1.2.1 Extraction of Genomic DNA

[0058] Tender leaves of 185 above-mentioned Chinese cherry resources were collected and dried and preserved with silica gel. After being taken back to the laboratory, the genomic DNA of each sample was extracted based on the improved CTAB method, and the DNA concentration and purity were detected by a nucleic acid protein analyzer, and the integrity of DNA was detected by 1% agarose gel electrophoresis.

[0059] 1.2.2 Whole Genome Resequencing and Filtering of Raw Reads

[0060] After fragmenting the total genomic DNA, a cDNA library was constructed through steps such as fragment size selection, DNA end repair, ligation of sequencing adapters, and PCR amplification. Whole-genome resequencing was performed based on the Illumina 2000 platform to obtain the original reads. The original reads were subjected to quality inspection in the Fastp software to remove adapters and low-quality reads, obtaining high-quality reads. Software parameters: --qualified_quality_phred 15 --unqualified_percent_limit 40 --n_base_limit 5 --detect_adapter_for_pe.

[0061] 1.2.3 SNP Variant Detection and Quality Control

[0062] Using the genome of Chinese cherry Luoyang ancient cherry (Cerasus pseudocerasus 'LuYg') as the reference genome (the genome data has been uploaded to the CNGB database, Accession Number: CNP0006741), the high-quality reads obtained in 1.2.2 were aligned to the reference genome in the BWA software, and then sorted and marked for repetitive sequences. Subsequently, variant detection was carried out using the GATK and Sentieon software to obtain the original gvcf file for each sample. Subsequently, based on the gvcf files of 185 samples, joint-genotype of the population was performed in GATK and Sentieon to obtain a vcf file containing the variant site information of all individuals. SNP sites were extracted from this vcf file and quality control was carried out. The quality control parameters were: -filter QD<2.0 -filterFS>60.0 -filter MQ<40.0 -filter MQRankSum<-12.5 -filter ReadPosRankSum<-8.0. Subsequently, in VCFtools, indicators such as allele frequency and sequencing depth of the SNP sites were further filtered to obtain 469,776 high-quality SNP sites for subsequent analysis. The filtering parameters were: --maf 0.01 --max-missing 0.9 --minDP 3 --minQ30 --min-alleles 2 --max-alleles 2 --hwe 0.001.

[0063] 1.2.4 Population Genetics Analysis and Construction of DNA Fingerprint

[0064] The high-quality SNP loci obtained in 1.2.3 were further screened based on the following principles to obtain core marker loci: (i) the SNP marker loci were evenly distributed on eight linkage groups (chr1-chr8); (ii) there was no missing data for all SNP marker loci; (iii) the linkage disequilibrium decay (LD decay) value of Chinese cherry at the species level calculated based on 469,776 SNP marker loci was 50 bp, so there were no other SNP mutation sites within 50 bp before and after the retained marker loci. Finally, 1,674 core SNP molecular markers of Chinese cherry resources were obtained. Based on the aforementioned 469,776 SNP molecular markers and the 1,674 core SNP molecular markers screened out, the population genetic structure of Chinese cherry was detected in the ADMIXTURE software (K = 1-20), principal component analysis (PCA) was performed using the EIGENSOFT software, the genetic distance between individuals was calculated in VCF2Dis, and the final neighbor-joining method (NJ) clustering tree was generated using phylip. Finally, the 1,674 core SNP molecular marker loci were sorted according to their chromosomal and physical positions, and a DNA fingerprint map specific to each individual was constructed according to the mutation type and genotype, where each row represents a sample and each column represents an SNP locus. The homozygous loci of the four types of nucleotides A, C, G, and T and all types of heterozygous loci are represented by five different colors from black to light gray, and the color codes are "#040000", "#404041", "#7F7F7F", "#C0BFBF", and "#EBEBEB" in turn. At the same time, the homozygous loci identical to the reference genome are represented by "0", the homozygous mutation loci are represented by "1", and the heterozygous loci are represented by "2" to generate the fingerprint code of each individual.

[0065] 2. Results

[0066] 2.1 Whole-genome resequencing, locus filtering, and discovery of high-quality SNP molecular marker loci

[0067] Whole-genome resequencing of 185 Chinese cherry resources generated approximately 350 Gb of raw sequencing data, with an average Q20 of 94.06%, an average Q30 of 87.20%, and an average GC content of 39.39%. After filtering the raw data with Fastp, the average Q20 increased to 96.81%, the average Q30 increased to 90.72%, and the average GC content was 39.16%. The high-quality reads of the 185 individuals after filtering were aligned with the Chinese cherry reference genome, with an average sequencing depth of 5× and an average coverage of 87.87%. After filtering with GATK and VCFtools, a total of 469,776 SNP loci were retained. These loci did not deviate from the Hardy-Weinberg equilibrium, with a minimum sequencing depth of 3, a minimum allele frequency of 0.01, and were bi-allelic loci, suitable for various population genetics studies. Subsequently, these 469,776 SNP loci were further filtered on the principle of not including missing data and a physical position difference of adjacent SNPs greater than 50 bp. Finally, 1,674 SNP molecular marker loci were screened out, which were evenly distributed on the chr1-chr8 linkage groups ( Figure 1 ), and these 1,674 SNP loci were the core SNP molecular marker loci of the 185 cherry resources.

[0068] 2.2 Evaluation of core SNP molecular marker loci

[0069] To evaluate the reliability and representativeness of the 1,674 core SNP molecular marker loci screened and discovered in DNA fingerprint construction and other work, we simultaneously used 469,776 SNP loci and 1,674 core SNP loci to detect the population genetic structure of 185 Chinese cherry resources ( Figure 2 ), PCA analysis ( Figure 3 ), and NJ phylogenetic tree construction ( Figure 4 ). The results showed that the results of the 1,674 core SNP molecular marker loci in the above three analyses were highly consistent with those of the 469,776 SNP loci analysis, indicating that the 1,674 core SNP molecular marker loci developed in this application could truly reflect the population genetic structure and genetic relationship of Chinese cherry germplasm resources, and the DNA fingerprint constructed with them could more comprehensively and truly reflect the core characteristics of the genetic information and genetic differences of Chinese cherry germplasm resources.

[0070] 2.3 Construction of DNA fingerprints of Chinese cherry resources

[0071] Figure 2It can be seen that when K = 3, Chinese cherry resources have three gene pools, of which wild Chinese cherry is a separate gene pool. Chinese cherry local germplasm is divided into two main gene pools, and the gene pools are divided by individuals with a Q value ≥ 0.5 as the threshold. One gene pool contains almost all individuals in the North China and East China Plain and some individuals in the Qinling Mountains and the Yunnan-Guizhou Plateau. The other gene pool contains most individuals in the Longmenshan seismic zone and the remaining individuals in the Qinling Mountains and the Yunnan-Guizhou Plateau. Compared with the germplasm resources in the North China and East China Plain and the Longmenshan seismic zone, the gene pools of the Qinling Mountains and the Yunnan-Guizhou Plateau germplasm resources are relatively mixed. In addition, individuals in the Longmenshan seismic zone, the Qinling Mountains and the Yunnan-Guizhou Plateau all show more frequent gene exchanges with wild Chinese cherries, which is consistent with the natural distribution area of ​​wild Chinese cherries. Figure 3 and Figure 4 These are the PCA analysis and NJ clustering results of the genetic relationships of 185 cherry resources. Figure 2 , Figure 3 and Figure 4 The results show that the population genetic structure based on 1674 core SNP molecular markers and 469776 SNP loci in 185 cherry resources ( Figure 2 ), PCA results shown in genetic relationships ( Figure 3 ) and NJ clustering tree topology ( Figure 4 ) were highly consistent, reflecting that the 1674 core SNP molecular marker loci were accurate and reliable in identifying the genetic structure and genetic relationship of Chinese cherry resource populations.

[0072] After confirming the representativeness of the core SNP molecular markers, we sorted and compared the 1674 core SNP molecular marker sites selected from the aforementioned 469776 SNP sites according to their physical positions, and further used these 1674 core SNP molecular marker sites to generate fingerprint sequences for each individual (Table 3), and realized the DNA fingerprint of 185 Chinese cherry resources ( Figure 5 ). This map can be used to efficiently evaluate the DNA genetic information and genetic differences of Chinese cherry resources, and provide a scientific basis for molecular-assisted selection in Chinese cherry breeding.

[0073] Table 3 DNA fingerprint sequences of 185 Chinese cherry resources

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[0090]

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[0094]

[0095] The specific embodiments described above further elaborate on the objectives, technical solutions, and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A core SNP molecular marker locus composition of Chinese cherry [Cerasus pseudocerasus (Lindl.) G.Don] resources, characterized in that: The SNP molecular marker site composition is composed of 1674 SNP molecular marker sites distributed on chr1 to chr8, and the SNP site information is shown in the following table; taking SNP1 as an example, the type G\A indicates that the reference genome of the site is G, the variable site type is A, and the rest of the sites are similar; The physical positions of the above-mentioned SNP molecular markers are based on the genome of the Chinese cherry Luoyang ancient cherry (Cerasus pseudocerasus'LuYg') as the reference genome and have been uploaded to the CNGB database (Accession Number: CNP0006741).

2. A SNP molecular marker composition comprising the Chinese cherry [Cerasus pseudocerasus (Lindl.) G.Don] resource core SNP molecular marker site composition according to claim 1.

3. A DNA fingerprint comprising the core SNP molecular marker site composition of the Chinese cherry [Cerasus pseudocerasus (Lindl.) G.Don] resource according to claim 1.

4. A DNA fingerprint database comprising the core SNP molecular marker site composition of Chinese cherry [Cerasus pseudocerasus (Lindl.) G.Don] resources according to claim 1.

5. The method for constructing a DNA fingerprint database according to claim 4, comprising the following steps: (1) Genomic DNA extraction: Extract the whole genome DNA of the sample to be tested; (2) Whole genome resequencing: Whole genome resequencing is performed on the genomic DNA obtained in step (1) to obtain double-end reads; (3) Raw reads filtering: Perform data filtering on the paired-end reads obtained in step (2) to obtain high-quality reads; (4) SNP detection: The high-quality reads obtained in step (3) are compared with the reference genome to perform variation detection and SNP marker site screening; (5) High-quality SNP marker discovery: The SNP marker sites obtained in step (4) are filtered according to the sequencing depth, missing data, site type, whether they deviate from Hardy-Weinberg equilibrium, etc., to obtain the SNP marker sites after secondary screening; (6) Evaluation of core SNP molecular marker sites and construction of DNA fingerprint map: The SNP marker sites obtained in step (5) were further filtered according to the missing sites and physical positions to obtain the core SNP molecular marker site combination, which was used to detect the population genetic structure of Chinese cherry resources, conduct principal component analysis (PCA), construct a cluster tree, clarify the kinship relationship, and build a DNA fingerprint map database.

6. The method for constructing a DNA fingerprint database as claimed in claim 5, wherein in step (1), a modified CTAB method is used to extract genomic DNA from the sample to be tested, the DNA concentration and purity are detected in a nucleic acid protein analyzer, and the integrity of the DNA is detected by 1% agarose gel electrophoresis; In step (2), a cDNA library is constructed and whole genome resequencing is performed based on the Illumina 2000 platform to obtain raw reads; In step (3), the original reads obtained in step (2) are quality checked in Fastp software to remove the adapters and low-quality reads to obtain high-quality reads; In step (4), the high-quality reads obtained in step (3) were aligned to the reference genome in the BWA software, and sorted and repeated sequences were marked. Then, GATK and Sentieon software were used to perform variation detection, and the original gvcf file was obtained. SNP sites were extracted from it for quality control. The quality control parameters were: -filter QD<2.0-filter FS>60.0-filter MQ<40.0-filter MQRankSum<-12.5-filter ReadPosRankSum<-8.0; The method for discovering high-quality SNP marker sites in step (5) is as follows: In VCFtools software, the SNPs obtained in step (4) are strictly screened according to the sequencing depth, missing data, site type, whether they deviate from Hardy-Weinberg equilibrium and other conditions, and 469,776 SNP marker sites are obtained after secondary screening. The specific parameters are: --maf 0.01 --max-missing 0.9 --mac 3 --minDP 3 --minQ 30 --min-alleles 2 --max-alleles 2 --hwe 0.001; The method for obtaining the core SNP molecular marker site combination in step (6) is as follows: (i) the SNP marker sites are evenly distributed on eight linkage groups (chr1 to chr8); (ii) there is no missing data in all SNP marker sites; (iii) the linkage disequilibrium decay (LD decay) value of the Chinese cherry species level calculated based on 469,776 SNP marker sites is 50 bp, so there are no other SNP mutation sites within 50 bp before and after the retained marker sites, and 1,674 core SNP molecular marker site combinations are obtained to carry out population genetic structure detection, principal component and cluster tree analysis of Chinese cherry resources, and construct a DNA fingerprint map database.

7. The method for constructing a DNA fingerprint database as described in claim 6, wherein in step (iii), based on the aforementioned 469776 SNP molecular marker sites and the 1674 core SNP molecular marker sites selected therefrom, the detection of the genetic structure of the Chinese cherry resource population is carried out in the ADMIXTURE software, the principal component analysis PCA is performed using the EIGENSOFT software, the genetic distance between individuals is calculated in VCF2Dis and the final neighbor-joining method, NJ clustering tree is generated using phylip, and finally, the 1674 core SNP molecular marker sites are sorted and arranged according to the chromosome and physical position of the site, and a specific DNA fingerprint map of each individual is constructed according to the variation type and genotype.

8. The method for constructing a DNA fingerprint database as claimed in claim 5, wherein the sample to be tested in step (1) is young leaves.

9. The SNP molecular marker site composition according to claim 1, the SNP molecular marker composition according to claim 2, and the DNA fingerprint map are used in (A) identifying Chinese cherry resources; (B) evaluating DNA genetic information and genetic differences of Chinese cherry resources; (C) analyzing the kinship, population genetic structure and genetic diversity of Chinese cherry resources; (D) constructing a Chinese cherry core germplasm and DNA fingerprint map database; (E) one or more aspects of molecular assisted selection in Chinese cherry breeding.

10. The DNA fingerprint database of claim 4 is used in one or more aspects of (a) identifying Chinese cherry resources; (b) evaluating DNA genetic information and genetic differences of Chinese cherry resources; (c) analyzing the kinship, population genetic structure and genetic diversity of Chinese cherry resources; and (d) molecular assisted selection of Chinese cherry breeding.

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