SNP molecular marker combination for constructing DNA fingerprint of genus Corylus and application thereof

Abstract

The application discloses a SNP molecular marker combination for constructing a DNA fingerprint of Corylus and application thereof. The SNP molecular marker combination of the application comprises 50 SNP molecular markers, can be applied to construction of a DNA fingerprint of Corylus and further applied to variety identification of Corylus, fills a blank of SNP molecular markers of Corylus and the DNA fingerprint of Corylus based on the SNP molecular markers, and by using DNA fingerprint information of different Corylus varieties, Corylus germplasm resources can be accurately and efficiently distinguished and identified, and the application has important significance for development and protection of the Corylus germplasm resources.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of molecular biology technology, and particularly relates to a SNP molecular marker combination for constructing a DNA fingerprint of Corylus and application thereof. BACKGROUND

[0002] Corylus plants belong to the Coryleae tribe of the Betulaceae family, and there are about 20 species in the genus, which are distributed in Asia, Europe and North America. There are 13 wild species widely recognized in the world. The widely cultivated hazelnut varieties abroad are C. avellana L. and its hybrid offspring with C. maxima Mill., and the main varieties include Tombul (Turkey), TGDL (Italy), Barcelona (USA), Jefferson (USA) and the like.

[0003] China is one of the original habitats of Corylus plants, and there are 8 species and 2 varieties, more than 90% of which are wild C. heterophylla Fisch. The main cultivation is C. heterophylla Fisch. and a small amount of garden cultivation of C. avellana L. C. heterophylla Fisch. is a hybrid offspring obtained by interspecific hybridization of C. heterophylla Fisch. and C. avellana L. in China in the 1980s. In the past 40 years, more than 10 varieties and more than 30 excellent lines have been identified and screened, which have the characteristics of large fruit, high yield, high kernel rate, full kernel, strong adaptability and the like, and are widely introduced to all parts of the country. At present, the cultivation area is nearly 1.2 million mu in China.

[0004] With the rapid development of C. heterophylla Fisch. industry, there are some problems to be solved in scientific research and industry. First, the existing C. heterophylla Fisch. varieties (lines) are screened from a mixed offspring population of multiple maternal and paternal parents, and the genetic relationship between the varieties (lines) is very complex, and some varieties (lines) are difficult to distinguish. Second, hybridization breeding is the main means of hazelnut germplasm innovation, and the hybridization breeding work of each unit has been continuously carried out, and the parentage identification work of the candidate population is urgently needed. Third, new varieties are being identified by each unit, and the identification of new varieties urgently needs precise and fast technology. Fourth, the problem of mixed varieties and different names of the same thing of C. heterophylla Fisch. seedlings is particularly common in production, and the mixed varieties or poor adaptability or incompatibility with pollination varieties all lead to huge economic losses of the planters.

[0005] Therefore, simple, precise and fast variety identification technology is urgently needed to be applied to the production and scientific research of Corylus varieties.

[0006] DNA fingerprint is composed of a small number of highly representative markers, which can reflect the individual differences of organisms, distinguish different individuals in the same species, and has high individual specificity and stability. It is an effective means for convenient, accurate and stable identification of germplasm resources and varieties, and is widely used in crop germplasm research. At present, domestic scholars have successively used RAPD, ISSR and AFLP techniques to study the genetic relationship of Corylus heterophylla x Corylus avellana hybrids. Liu Zhenpan et al. used RAPD marker technology to conduct fingerprint analysis on 8 main hybrid hazelnut varieties in Liaoning, and constructed the fingerprint map of excellent hazelnut varieties. Chen Xin et al. used ISSR molecular marker method to study the genetic relationship of 17 main Corylus heterophylla x Corylus avellana hybrid varieties (lines). Ma Qinghua et al. used AFLP analysis on 10 main varieties such as Dawei, and screened 15 pairs of Pst I / Mse I primers with strong polymorphism from 64 pairs of primers, and obtained 1739 bands, which can be used for rapid identification of main Corylus heterophylla x Corylus avellana hybrid varieties. However, the above-mentioned molecular marker methods have problems of time-consuming, low efficiency, poor accuracy, poor universality and the like, and have not realized digital comparison. Chinese invention patent ZL201710385371.X discloses a method for identifying Corylus heterophylla x Corylus avellana hybrid varieties by using SSR markers. The technology uses SSR molecular markers combined with capillary electrophoresis technology to establish a Corylus heterophylla x Corylus avellana hybrid variety identification method based on 4 pairs of EST-SSR markers. However, the detection range of the method is limited to a limited number of varieties, the universality and the distinction of similar varieties are not decisive, and the cost consumption is large.

[0007] SNP marker, i.e. single nucleotide polymorphism, refers to the variation of a single nucleotide on the genome, including substitution, transversion, deletion and insertion, which forms a genetic marker. SNP marker has the advantages of large number, wide distribution, high genetic stability, strong representation, easy genotyping and rapid and efficient detection, and is widely used in the construction of core germplasm and fingerprint map of crops such as rice, corn, sorghum, tea tree and soybean. It provides important resources for crop breeding, genome research and genetic research, and plays an important role in crop variety identification, purity determination and variety right protection. Therefore, it is of great significance to use SNP sites to construct DNA fingerprint map and identify hazelnut germplasm for the development and protection of hazelnut germplasm resources. SUMMARY

[0008] The present application provides a SNP molecular marker combination for constructing hazel DNA fingerprint map and its application, which provides a basis for the development and protection of hazel plant germplasm resources.

[0009] The specific technical solutions are as follows:

[0010] One of the purposes of the present application is to provide a SNP molecular marker combination for constructing a DNA fingerprint of Corylus, which comprises 50 SNP molecular markers, the site information corresponding to the SNP molecular markers is shown in Table 1, and the SNP molecular markers are numbered as SNP1-SNP50.

[0011] Table 1 Site information corresponding to the SNP molecular markers of Corylus

[0012]

[0013]

[0014] Specifically, the above-mentioned SNP molecular marker combination is determined based on the reference genome of the female parent flat hazel, and the genome version number is GCA_016403345.1.

[0015] The second purpose of the present application is to provide the application of the above-mentioned SNP molecular marker combination in constructing the DNA fingerprint of Corylus. The present application takes 37 Corylus plant variety resources as materials, applies the above-mentioned SNP molecular marker combination, and establishes the DNA fingerprint of Corylus.

[0016] The third purpose of the present application is to provide the application of the above-mentioned SNP molecular marker combination in the identification of Corylus plant varieties. The DNA fingerprint of Corylus established by applying the above-mentioned SNP molecular marker combination can be used for variety identification by comparing the genotypes of SNP sites of the sample to be tested with the fingerprint, so as to know whether the sample to be tested is a suspected same variety or a different variety of a certain standard variety in the DNA fingerprint of Corylus. The identification standard is that the number of interspecific difference sites is greater than or equal to 2, which is a different variety, and the number of interspecific difference sites is less than or equal to 1, which is a suspected same variety.

[0017] The fourth purpose of the present application is to provide a construction method of a DNA fingerprint of Corylus, which comprises the following steps:

[0018] Obtaining whole genome resequencing data of a plurality of Corylus variety samples, and obtaining the genotypes of each SNP molecular marker in the above-mentioned SNP molecular marker combination by comparing with a reference genome, so as to construct the DNA fingerprint of Corylus.

[0019] More specifically, the construction method is as follows: extracting the DNA of the Corylus variety resources used for constructing the DNA fingerprint, detecting the genotypes of the 50 SNP sites by resequencing; arranging the genotypes of each Corylus variety at the above-mentioned 50 SNP sites in order according to the site number, forming a specific sequence, and the specific sequence is the DNA fingerprint of Corylus.

[0020] The fifth object of the present application is to provide a DNA fingerprint of Corylus obtained by using the above construction method.

[0021] The sixth object of the present application is to provide an application of the above DNA fingerprint of Corylus in the identification of Corylus plant varieties.

[0022] The beneficial effects of the present application are as follows:

[0023] The present application develops a SNP molecular marker combination of Corylus plants comprising 50 SNP molecular markers. The SNP molecular marker combination can be applied to the construction of a DNA fingerprint of Corylus plants and further applied to the identification of Corylus plant varieties, filling the blank of SNP molecular markers of Corylus plants and DNA fingerprints of Corylus plants based on SNP molecular markers. Using the DNA fingerprint information of different Corylus varieties, Corylus plant variety resources can be accurately and efficiently distinguished and identified, which has important significance for the development and protection of Corylus plant germplasm resources. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is a SNP distribution map of Example 1 with medium heterozygosity;

[0025] Figure 2 is a phylogenetic tree of 37 Corylus plant varieties constructed based on whole genome SNP molecular markers in Example 1;

[0026] Figure 3 is a phylogenetic tree of 37 Corylus plant varieties constructed based on the combination of 50 core SNP sites screened in Example 1;

[0027] Figure 4 is a location map of 50 SNP sites on chromosomes in Example 1;

[0028] Figure 5 is a SNP number distribution map within a 1Mb window size in Example 1;

[0029] Figure 6 is a heat map of sample genotype data of 37 Corylus plants. DETAILED DESCRIPTION

[0030] The principles and characteristics of the present application are described below in combination with examples, which are used to explain the present application and are not used to limit the scope of the present application.

[0031] Example 1

[0032] The screening of the SNP molecular marker combination of Corylus plants is carried out, and the method steps are as follows:

[0033] S1. Selection of test samples: The maternal flat-hazelnut genome (GCA_016403345.1) was used as the reference genome, and 37 hazelnut varieties were used as samples. The varieties and source information of various germplasm resources are shown in Table 2.

[0034] Table 2 Varieties and sources of 37 hazelnut plants

[0035]

[0036]

[0037] S2. SNP identification: Genomic DNA was extracted from each hazelnut variety sample, and whole-genome resequencing was performed. BWA software (Burrows-Wheeler Aligner) was used to align the sequencing reads to the reference genome, and GATK (Genome Analysis Toolkit) was used to identify 9,440,145 SNPs.

[0038] S3. Screening of identified SNPs:

[0039] (1) Basic quality control filtering was performed using bcftools and vcftools software: low-quality sites (QUAL < 30), sites with high deletion rates (deletion rate > 10%), and sites with low minor allele frequency (MAF) (MAF < 5%) were removed;

[0040] (2) Double-allele SNP sites were screened using bcftools software: only double-allele SNP sites were retained;

[0041] (3) Linkage disequilibrium (LD) pruning: plink software was used for LD pruning (r2 < 0.2, window size 500 kb, step size 50), and LD-pruned SNPs were extracted;

[0042] (4) High polymorphic sites (heterozygosity) were screened using plink software: the observed heterozygosity of each SNP site was calculated, and medium heterozygosity sites (Het value 0.3-0.7) were screened; the distribution of medium heterozygosity SNPs is shown in Figure 1 ;

[0043] (5) Uniform distribution and representative screening: SNP density was controlled to avoid excessive density or sparseness, and the —thin function of vcftools software was used to set the interval; finally, 50 core SNPs were screened, which constituted the hazelnut SNP molecular marker combination, as shown in Table 1.

[0044] S4. Identification of the effectiveness of the obtained SNP sites:

[0045] The phylogenetic trees of 37 Corylus varieties were constructed based on the sequencing obtained whole genome SNP molecular markers and the screened 50 core SNP site combinations, respectively. The phylogenetic tree of 37 Corylus varieties constructed based on the whole genome SNP molecular markers is shown in Figure 2 ; the phylogenetic tree of 37 Corylus varieties constructed based on the screened 50 core SNP site combinations is shown in Figure 3 . It can be seen by comparison that only a few germplasms have different clustering relationships on the two evolution trees, which may be related to the existence of convergent genetic variation among these germplasms due to the existence of hybrid gene flow. Overall, the 50 core SNP markers can effectively distinguish each Corylus variety. The obtained 50 core SNPs are uniformly distributed on the 11 chromosomes of the Corylus plant genome; the positions of the 50 SNP markers on the 11 chromosomes of the Corylus plant are shown in Figure 4 ; the SNP number distribution within a 1 Mb window is shown in Figure 5 .

[0046] Example 2

[0047] Corylus DNA fingerprinting was performed: based on the reference gene sites of each SNP in the SNP molecular marker combination obtained in Example 1, the genotype of each sample was extracted and converted to base representation, and the genotype of each Corylus variety at the above-mentioned 50 SNP sites was combined in order according to the site number to form a specific sequence, and the DNA fingerprint of 37 Corylus plants was obtained, as shown in Tables 3-6. Figure 6 The genotype data heat map of 37 Corylus plant samples. Figure 6 In the figure, the horizontal coordinate is the Corylus plant variety, and the vertical coordinate is the SNP molecular marker number (the number n on the vertical coordinate corresponds to the molecular marker number SNPn, n = 1-50).

[0048] Table 3 Corylus DNA fingerprint (serial numbers 1-10)

[0049]

[0050]

[0051] Table 4 Corylus DNA fingerprint (serial numbers 11-20)

[0052]

[0053]

[0054] Table 5 Corylus DNA fingerprint (serial numbers 21-30)

[0055]

[0056]

[0057] Table 6 DNA fingerprint of Corylus (No. 31-37)

[0058]

[0059]

[0060] The above-mentioned DNA fingerprint of Corylus can be used for the identification of Corylus varieties. The identification method is as follows: the DNA of the Corylus to be identified is extracted and subjected to whole genome sequencing, the genotypes of the above-mentioned 50 SNP sites are detected, and the above-mentioned DNA fingerprint is compared to perform variety identification. The identification standard is: the number of interspecific difference sites is ≥2 for different varieties, and the number of interspecific difference sites is ≤1 for suspected same varieties.

[0061] The above merely provides the preferred embodiments of the present application, and is not used to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A method for constructing a DNA fingerprint of the genus *Corylus*, characterized in that, Includes the following steps: Whole-genome resequencing data of multiple hazelnut species samples were obtained, and the genotypes of each SNP molecular marker in the SNP molecular marker combination shown in the table below were obtained, thereby constructing a hazelnut DNA fingerprint map. The reference genome of the SNP molecular markers is the maternal parent hazelnut, with the genome version number GCA_016403345.

1. 。 2. An application of a Hazelnut DNA fingerprint constructed using the construction method described in claim 1 in the identification of Hazelnut plant varieties.

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