Mulberry whole genome liquid phase chip and application thereof

Abstract

The invention discloses a mulberry whole genome liquid phase chip. Genetic typing sites of the liquid phase chip comprise 45668 SNP (Single Nucleotide Polymorphism) sites which are positioned on a mulberry reference genome MorusalbaASM 1206604v3 in total. According to the mulberry whole genome liquid phase chip, 45668 SNP loci capable of being used for chip design are screened out from large-scale sequencing data, the loci are sourced from more mulberry varieties in China, and the universality is higher; the mulberry whole genome chip disclosed by the invention is suitable for a targeted capture sequencing technology, not only can be used for typing probe targets, but also can be used for typing amplification areas around the probe targets through corresponding probe design, so that compared with a solid-phase chip with the same number of probes, the mulberry whole genome chip disclosed by the invention can be used for detecting more variation sites; meanwhile, the typing cost is obviously reduced on the basis of increasing the number of detection sites.

Description

Technical Field

[0001] The invention belongs to the technical field of gene chips, relates to a mulberry whole genome liquid phase chip, and also relates to the application of the mulberry whole genome liquid phase chip. Background Art

[0002] DNA molecular markers are genetic markers based on nucleotide sequence variation between individuals and are an important basis for molecular breeding. Single nucleotide polymorphisms (SNPs) are formed by variations in a single nucleotide in the genome, such as substitution, transversion, insertion or deletion. SNPs are the most common molecular markers because of their large number and high detection accuracy.

[0003] At present, the main methods for SNP site typing are whole genome resequencing and gene chips. Whole genome resequencing is to randomly interrupt DNA at the level of the entire genome, then add adapters to the fragmented samples, and sequence the inserted fragments after amplification. Although the cost of single sample sequencing has been significantly reduced, whole genome sequencing for hundreds or thousands of samples still faces huge challenges of high cost and massive data processing. In order to reduce costs and data volume, gene chips based on typing of partial sites or regions have emerged and are widely used, and high-density molecular markers covering the entire genome can be detected with a smaller sequencing volume. Gene chips mainly include solid-phase chips and liquid-phase chips. Solid-phase chips identify gene information based on the fluorescent color signal after hybridization of the marker sequence on the chip with the genome; liquid-phase chips use biotin-labeled probes designed for the area near the target, and after hybridization with the genome, amplification, library construction, and second-generation sequencing are performed to achieve typing of key regions of the genome. Compared with solid-phase chips, liquid-phase chips have the following advantages: Based on the principle of amplification, liquid-phase chips can not only type the target site, but also other sites around the target site within the probe coverage range; secondly, the detection sites of liquid-phase chips are more flexible and the typing cost is lower. Currently, liquid-phase chips used in mulberry-related fields still have the disadvantage of low sequencing data coverage.

[0004] In summary, the existing technology has the problem of low coverage of mulberry whole genome liquid phase chip sequencing data. Summary of the invention

[0005] The purpose of the present invention is to provide a mulberry whole genome liquid phase chip, which solves the problem of low coverage of mulberry whole genome liquid phase chip sequencing data in the prior art.

[0006] The technical solution adopted by the present invention is a mulberry whole genome liquid phase chip, the genotyping sites of the liquid phase chip include a total of 45668 SNP sites located on the mulberry reference genome Morus_alba_ASM1206604v3, the deletion rate of the 45668 SNP sites is not more than 10%, the heterozygosity rate is not more than 50%, and the minimum allele variation frequency is not less than 0.3.

[0007] The present invention is also characterized in that: the liquid phase chip also includes a probe sequence covering the SNP site corresponding to the mulberry reference gene sequence, the length of the probe sequence is 110bp, the GC content is between 30% and 80%, and the number of homology regions is ≤5; the position information of 45668 SNP sites is shown in Table 1, wherein Chr1 in Chr1_11625171 indicates that the SNP site is located on chromosome 1 of the mulberry genome, 11625171 is the specific position of the SNP site on the chromosome, which is 11625171bp, and the remaining SNP sites are similar.

[0008] The present invention also provides:

[0009] Application of mulberry whole genome liquid phase chip in genetic diversity analysis of mulberry varieties.

[0010] Application of mulberry whole genome liquid phase chip in mulberry variety identification.

[0011] Application of mulberry whole genome liquid phase chip in identification of mulberry genetic relationships.

[0012] Application of mulberry whole genome liquid phase chip in the evaluation and improvement of mulberry germplasm resources.

[0013] Application of mulberry whole-genome liquid phase array in mulberry whole-genome association analysis.

[0014] Application of mulberry whole genome liquid phase chip in mulberry whole genome selection breeding.

[0015] Application of whole genome liquid phase microarray in background selection of mulberry based on molecular markers.

[0016] Application of mulberry whole genome liquid phase chip in DNA fingerprint identification.

[0017] The beneficial effects of the present invention are as follows: the present invention screens out 45668 SNP sites that can be used for chip design from large-scale sequencing data, and the sources of the sites cover more domestic mulberry varieties, and the versatility is stronger; the mulberry whole genome chip of the present invention is suitable for targeted capture sequencing technology, and through corresponding probe design, not only can the probe target point be typed, but also the amplification area around the probe target point can be typed. Therefore, compared with a solid phase chip with the same number of probes, more variant sites can be detected, and the typing cost is significantly reduced on the basis of increasing the number of detection sites. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The mulberry whole genome liquid phase chip of the present invention includes a distribution diagram of SNP sites on the mulberry genome. DETAILED DESCRIPTION

[0019] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0020] The present invention provides a mulberry whole genome liquid phase chip, wherein the genotyping sites of the liquid phase chip include a total of 45668 SNP sites located on the mulberry reference genome Morus_alba_ASM1206604v3, wherein the deletion rate of the 45668 SNP sites is not greater than 10%, the heterozygosity rate is not greater than 50%, and the minimum allele variation frequency is not less than 0.3; Figure 1 Shown is the distribution of 45668 SNP loci on the mulberry genome;

[0021] The liquid phase chip also includes a probe sequence covering the SNP sites corresponding to the mulberry reference gene sequence. The probe sequence is 110 bp long, the GC content is between 30% and 80%, and the number of homology regions is ≤5. The position information of the 45,668 SNP sites is shown in Table 1:

[0022] Table 1 Location information of SNP sites

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[0070] Among them, Chr1 in Chr1_11625171 indicates that the SNP site is located on chromosome 1 of the mulberry genome, 11625171 is the specific position of the SNP site on the chromosome, which is 11625171 bp, and the same applies to the remaining SNP sites.

[0071] The preparation method of the mulberry whole genome liquid phase chip is as follows:

[0072] Step 1: Obtain sequencing data of mulberry varieties and screen chip sites based on the mulberry reference genome;

[0073] Acquire sequencing data of mulberry varieties, use FastQC software to remove low-quality and adapter-contaminated base sequences in the sequencing data of mulberry varieties to obtain clean sequencing data of mulberry varieties, use BWA software to align the clean sequencing data of mulberry varieties with the mulberry reference genome, use GATK software to combine the mulberry reference genome to perform variation detection on the clean sequencing data of mulberry varieties, and obtain chip sites after data filtering;

[0074] The chip loci include 45668 SNP loci with a deletion rate of no more than 10%, a heterozygosity rate of no more than 50%, a minimum allele variation frequency of no less than 0.3, and a uniform distribution on the chromosome. The distribution of these SNP loci on the mulberry reference genome is as follows: Figure 1 As shown;

[0075] Step 2: Prepare a mulberry whole genome liquid phase chip based on the chip sites;

[0076] After the SNP site screening was completed, probe design was performed, and a mulberry 40K liquid phase targeted capture probe was obtained according to the principle that the probe length was 110bp, the probe GC content was between 30% and 80%, and the number of homology regions was ≤5; the liquid phase targeted capture probe was synthesized by single-stranded nucleotides according to the designed nucleotide sequence, and the two synthesized DNA nucleotide sequences with a length of 110bp and modified with a biotin group at the 5' end were mulberry liquid phase probes; the synthesized mulberry liquid phase probes were mixed with equimolar masses, and the volume was fixed to 3pmol / mL using a mixed solution of EDTA and Tris HCl to obtain a mulberry whole genome liquid phase chip probe mixture.

[0077] The mulberry whole genome liquid phase chip of the present invention has the following characteristics: high detection efficiency: the liquid phase chip of the present invention can detect more SNP sites, the detection reagent cost is significantly reduced, the cost price advantage of single marker detection is more obvious, and large-scale typing detection of mulberry trees can be performed; flexibility of chip upgrade: the liquid phase chip of the present invention has no sample amount limit for a single detection, and there is no need to adjust the existing probes when adding new probes in the later stage, thereby ensuring the flexibility of upgrading; wide application: the samples used for designing the mulberry liquid phase chip in the present invention are derived from the most representative materials in China, covering wild materials and cultivated varieties, with wide varieties distribution and strong representativeness; it is conducive to the development of genome selection breeding, germplasm resource evaluation, DNA fingerprint identification and related research on GWAS association analysis.

[0078] Example 1

[0079] This embodiment proposes a mulberry whole genome liquid phase chip, the genotyping sites of the liquid phase chip include a total of 45668 SNP sites located on the mulberry reference genome Morus_alba_ASM1206604v3, the 45668 SNP sites have a deletion rate of no more than 10%, a heterozygosity rate of no more than 50%, and a minimum allele variation frequency of no less than 0.3. The liquid phase chip also includes a probe sequence covering the SNP site corresponding to the mulberry reference gene sequence, the probe sequence has a length of 110bp, a GC content between 30% and 80%, and a number of homology regions ≤5.

[0080] Example 2

[0081] This example proposes the application of mulberry whole genome liquid phase chip in the genetic similarity analysis of mulberry varieties, including the following steps:

[0082] (1) Extracting DNA from mulberry leaf samples and constructing a sample sequencing library; adding a mulberry 40K probe labeled with biotin, and hybridizing the probe with the target region of the genome to form a double strand;

[0083] (2) Using streptavidin-coated magnetic beads to molecularly adsorb the target probe labeled with biotin, thereby capturing the genomic fragments hybridized with the probe;

[0084] (3) Eluting, amplifying, building a library, and sequencing the captured target sequence to ultimately obtain the genotype of the target SNP site of the mulberry material;

[0085] (4) The genetic similarity between the mulberry materials was calculated based on the genotype results. The calculation formula is as follows:

[0086]

[0087] Where GS is the genetic similarity between sample 1 and sample 2; n ij N is the number of SNP marker sites detected in both sample 1 and sample 2 but with no genotype difference; ij is the number of SNP marker sites detected in both sample 1 and sample 2;

[0088] (5) Based on the calculated genetic similarity, determine the genetic similarity between different pineapple varieties.

[0089] Example 3

[0090] This example proposes the application of mulberry whole genome liquid phase chip in variety identification, including the following steps:

[0091] The method in Example 1 is used to perform loci typing on the varieties to be identified, and the genetic similarity between the varieties is calculated. The determination is made based on the genetic similarity results, and the determination method is as follows:

[0092] When the genetic similarity GS between the tested variety and all database varieties is no more than 90%, the tested variety is determined to be a new variety.

[0093] When the genetic similarity GS between the tested variety and any one of the database varieties is greater than 96%, the tested variety is determined to be a duplicate variety.

[0094] When the genetic similarity GS between the tested variety and all database varieties is between 90% and 96%, the tested variety is determined to be a similar variety.

Claims

1. A mulberry whole genome liquid phase chip, characterized in that: The genotyping sites of the liquid phase chip include a total of 45,668 SNP sites located on the mulberry reference genome Morus_alba_ASM1206604v3, the deletion rate of the 45,668 SNP sites is no more than 10%, the heterozygosity rate is no more than 50%, and the minimum allele variation frequency is no less than 0.

3.

2. The mulberry whole genome liquid phase chip according to claim 1, characterized in that: The liquid phase chip also includes a probe sequence covering the SNP sites corresponding to the mulberry reference gene sequence, the probe sequence is 110 bp in length, the GC content is between 30% and 80%, and the number of homology regions is ≤5; the position information of the 45,668 SNP sites is as follows: Among them, Chr1 in Chr1_11625171 indicates that the SNP site is located on chromosome 1 of the mulberry genome, 11625171 is the specific position of the SNP site on the chromosome, which is 11625171 bp, and the same applies to the remaining SNP sites.

3. Application of the mulberry whole genome liquid phase chip described in any one of claims 1-2 in the genetic diversity analysis of mulberry varieties.

4. Use of the mulberry whole genome liquid phase chip described in any one of claims 1-2 in mulberry variety identification.

5. Use of the mulberry whole genome liquid phase chip described in any one of claims 1-2 in identifying phylogenetic relationships of mulberry trees.

6. Application of the mulberry whole genome liquid phase chip according to any one of claims 1-2 in the evaluation and improvement of mulberry germplasm resources.

7. Use of the mulberry whole genome liquid phase chip described in any one of claims 1-2 in mulberry whole genome association analysis.

8. Use of the mulberry whole genome liquid phase chip according to any one of claims 1-2 in mulberry whole genome selection breeding.

9. Use of the mulberry whole genome liquid phase chip described in any one of claims 1-2 in background selection of mulberry based on molecular markers.

10. Use of the mulberry whole genome liquid phase chip according to any one of claims 1-2 in DNA fingerprint identification.

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