Ginkgo whole genome SNP site combination, liquid chip and application

Abstract

The invention discloses a ginkgo whole genome SNP site combination, a liquid phase chip and application, and belongs to the technical field of genomics, the ginkgo whole genome SNP site combination comprises 45776 SNP sites, and the physical positions of the 45776 SNP sites are determined based on ginkgo maleGinkgov3 reference genome comparison. The gingko whole genome liquid phase chip is a gingko 45K liquid phase chip based on targeted capture sequencing, and comprises a probe combination for detecting a gingko whole genome SNP (Single Nucleotide Polymorphism) site combination. According to the present invention, with the ginkgo whole genome SNP site combination and the liquid phase chip, the SNPs can be rapidly detected in the ginkgo individual to be detected so as to achieve the rapid genetic typing of the ginkgo, such that the method can be used for the geographical traceability analysis, the genetic relationship analysis, the sex determination and other scenes of the ginkgo germplasm resource.

Description

Technical Field

[0001] The invention belongs to the technical field of genomics, and specifically relates to a Ginkgo whole genome SNP site combination, a liquid phase chip and applications. Background Art

[0002] Ginkgo (Ginkgo biloba L.), also known as white fruit tree and maidenhair tree, is a tree species unique to my country. Ginkgo plants are tall and upright, with unique and beautiful leaf shape and color, and have long been loved by people; at the same time, it has unique edible, medicinal and timber value, and has broad development prospects and potential. As the origin of ginkgo, China has more than 90% of the world's ginkgo germplasm resources, but research still needs to be strengthened in the selection and breeding of ginkgo varieties or the development of new varieties. In addition, as a relic species with ancient origins, protecting wild germplasm resources is an important guarantee for maintaining the sustainable and healthy development of the ginkgo industry. The effective use of molecular markers is an important means to improve the level of protection of ginkgo germplasm resources and accelerate the progress of breeding.

[0003] Single nucleotide polymorphism (SNP) molecular markers are usually distributed throughout the genome, with a large number and high polymorphism, so they are widely used in biological genetics and related research. At present, the genotype detection of SNP markers is mainly based on genome resequencing or simplified genome sequencing. However, due to the large size of the Ginkgo genome (about 10.6Gb), the cost of deep resequencing is still high. First, the sequencing cost is high, and second, the computer computing power cost of multi-sample variation detection (SNP calling) of sequencing data is high. A feasible alternative is to use targeted capture technology to accurately locate and sequence the sequences of the target regions of the genome, reduce the sequencing coverage breadth and increase the sequencing depth, and obtain more reliable polymorphisms. The principle of targeted capture technology is to screen the target regions of the genome for sequencing typing, and then design characteristic probes for the target region sequences according to the base complementary pairing principle and thermodynamic stability algorithm, and then use the synthesized probes to hybridize with genomic DNA, capture and enrich the target region sequences on the genome, and finally perform second-generation sequencing and typing on the captured and enriched target regions, so as to obtain the genotypes of all SNP molecular markers in the target region. Compared with genome resequencing technology, the advantage of targeted capture technology is that it can amplify the signal of specific regions of the genome, obtain deeper coverage and higher data accuracy. Although it can only cover a limited range of the genome, this technology is suitable for in-depth research on specific regions or analysis of a large number of samples, especially for species with large genomes. At the same time, the results of targeted capture sequencing are highly repeatable. Even samples built at different times and in different batches have a repeatability of more than 99.9%, making the data cumulative and verifiable, saving sequencing and computing costs at multiple levels.

[0004] Gene chip is a comprehensive platform that can integrate probes of target regions, related reaction systems and genotyping processes. Among them, liquid phase chips can integrate tens of thousands of probes of target regions at a time, and new probes can be added at any time without adjusting the reaction system, which has the advantages of high efficiency and high flexibility. This technology was first used in medical fields such as human genetic disease detection and tumor screening. In recent years, species-specific gene chips have been developed for some common economic crops or model plants, such as rice, wheat, pear, tomato, corn, barley, broad beans and other species, and they have been used to detect SNPs and InDel sites in specific functional gene regions and regulatory regions, providing good data support for the study of molecular genetics, breeding improvement, population polymorphism, etc. of important traits (such as Chinese patent documents with publication numbers CN111684113A and CN118240958A, etc.). However, due to the low versatility of targeted probes, this rapid, efficient and automated genotyping method has not yet been practiced in most plants. Therefore, it is of great significance to design a whole-genome liquid phase chip suitable for Ginkgo to reduce the cost of obtaining SNP molecular markers, to reveal the genotype differences between Ginkgo individuals from different sources, and to study the association between genes and traits, so as to provide a scientific basis for protection and development. Summary of the invention

[0005] The present invention provides a ginkgo whole genome SNP site combination, a liquid phase chip and an application thereof. The present invention can be used to quickly detect SNPs in ginkgo individuals to be tested, thereby realizing rapid genotyping of ginkgo, and further can be used for scenarios such as geographic traceability analysis, kinship analysis, and gender identification of ginkgo germplasm resources.

[0006] The specific technical solutions adopted are as follows:

[0007] The present invention provides a ginkgo whole genome SNP site combination, the ginkgo whole genome SNP site combination includes 45776 SNP sites, the physical positions of the 45776 SNP sites are determined based on the comparison of the male_Ginkgo_v3 reference genome (https: / / ginkgo.zju.edu.cn / genome / ), and the information of the 45776 SNP sites is shown in Table 1, which is expressed in the form of chromosome: physical position (the reference genotype is published in the reference genome, and there are individual differences in variant allele genotypes).

[0008] The principles for screening SNP sites include: 1) screening sites that show high polymorphism in the resequencing results of the ginkgo population; 2) screening sites in the non-repetitive regions of the ginkgo genome; 3) including all polymorphic sites in the male-specific region (NRY) of the ginkgo genome; 4) in the autosomal region, try to make the SNP sites evenly distributed in the whole ginkgo genome.

[0009] Table 1 Information of 45776 SNP loci

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[0132] The present invention also designs a probe combination for detecting the SNP site combination of the whole genome of Ginkgo biloba, the probe is a double-stranded DNA probe, which is a nucleotide sequence designed and synthesized according to the screened SNP site. The probe is obtained according to the conventional liquid phase probe design principle in the prior art.

[0133] The present invention also provides the use of the Ginkgo whole genome SNP site combination in preparing the Ginkgo whole genome liquid phase chip.

[0134] The present invention also provides a ginkgo whole genome liquid phase chip, which is presented in the form of a liquid phase kit, comprising a probe combination for detecting the ginkgo whole genome SNP site combination and a reagent for targeted capture sequencing; the probe combination is used to identify the genotype of each SNP site in the ginkgo whole genome SNP site combination.

[0135] Furthermore, the liquid phase chip is a Ginkgo 45K liquid phase chip based on targeted capture sequencing.

[0136] The method for preparing the Ginkgo whole genome liquid phase chip comprises the following steps:

[0137] S1. Screening of high-quality SNP sites from the resequencing data of the Ginkgo population;

[0138] S2. Screening geographical lineage-specific, male-specific and highly polymorphic SNP loci from the above high-quality SNP loci;

[0139] S3. Remove the SNP sites that cannot design probes at both ends from the above sites;

[0140] S4. The above SNP sites were collected to prepare biotin-modified liquid phase probes, and finally a Ginkgo whole genome liquid phase chip was obtained.

[0141] The present invention also provides the application of the ginkgo whole genome SNP site combination, the probe combination for detecting the ginkgo whole genome SNP site combination or the ginkgo whole genome liquid phase chip in genetic diversity analysis, sex identification, geographical traceability analysis, kinship analysis, genotyping, gene positioning or molecular assisted breeding of ginkgo germplasm resources.

[0142] The present invention also provides a method for whole genome genotyping detection of Ginkgo biloba, comprising the following steps:

[0143] S1. Obtain genomic DNA of the ginkgo sample to be tested;

[0144] S2. Detecting the genomic DNA using the Ginkgo whole genome liquid phase chip to obtain raw data;

[0145] S3. Perform data analysis on the raw data to obtain genotyping results.

[0146] Compared with the prior art, the present invention has the following beneficial effects:

[0147] The present invention provides a ginkgo whole genome SNP site combination and a corresponding liquid phase chip. The ginkgo whole genome liquid phase chip of the present invention can quickly and accurately detect the genotype of the ginkgo individual to be tested, and the average SNP detection rate is 94.73%, and the average proportion of repeated sample detection and consistent genotyping results is 93.11%. The genotyping has good stability, high accuracy, and excellent overall performance. The ginkgo whole genome liquid phase chip prepared by the present invention is used to perform genotype detection on ginkgo samples, and the required sample amount is small, the sample number is unlimited, and the detection cost is low. In addition, the liquid phase chip contains sites related to gender and pedigree, which can be widely used for the accurate identification of ginkgo germplasm resources, providing important technical support for the protection of ginkgo resources and the cultivation of new varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0148] Figure 1 The invention provides the distribution of the Ginkgo whole genome SNP site combination on the genome.

[0149] Figure 2 This is a statistical graph of the capture rate of the Ginkgo whole genome 45K SNP liquid phase chip sites in different samples provided by the present invention.

[0150] Figure 3 This is a statistical graph of the consistency of genotypes detected by the Ginkgo whole genome 45K SNP liquid phase chip provided by the present invention in repeated samples. DETAILED DESCRIPTION

[0151] The present invention will be further described below in conjunction with the examples and accompanying drawings. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The operating methods of the following examples that do not specify specific conditions are usually performed under normal conditions or under conditions recommended by the manufacturer. The content that is not described in detail in this specification sheet belongs to the prior art known to professionals in the field. The experimental materials used in the following examples, if not otherwise specified, can be purchased by conventional biochemical reagent companies.

[0152] Example 1 Construction of Ginkgo Whole Genome 45K SNP Liquid Phase Chip

[0153] By collecting ginkgo from different geographical distributions around the world, the whole genome genetic information of 657 ginkgo individuals was obtained using whole genome resequencing technology. 53,363 SNP sites that met the standards were obtained through strict screening. SNP sites that could not design probes at both ends were removed from the above sites, and 45,776 capture probes were successfully synthesized. Finally, the ginkgo whole genome 45K SNP liquid phase chip was developed. The specific implementation steps are as follows:

[0154] (1) Whole-genome resequencing of Ginkgo biloba and identification and screening of SNP loci

[0155] Total DNA was extracted from 657 Ginkgo samples that had been collected and preserved. After the extracted DNA was qualified by agarose gel electrophoresis and Nanodrop detection, it was sequenced using whole genome resequencing technology. The quality control of the raw data (raw reads) was performed to remove reads with adapters, reads containing N greater than 5%, and low-quality reads to obtain clean reads. The clean reads were aligned with the Ginkgo reference genome (male_Ginkgo_v3, https: / / ginkgo.zju.edu.cn / genome / ) using BWA software, and then the GATK software was used to detect variant sites. The quality of the detected variant sites was screened, and sites with a minimum allele frequency <0.05, non-two-base sites, and sites deviating from the Hardy-Weinberg equilibrium were deleted. After filtering, about 16.03 million high-quality SNP sites were obtained.

[0156] (2) Development of a 45K SNP liquid phase array for the entire Ginkgo genome

[0157] On the basis of the above high-quality SNP sites, further strict screening was carried out. The screening principles were as follows: 1) Screening sites that showed high polymorphism in the resequencing results of the ginkgo population; 2) Screening sites located in the non-repetitive region of the ginkgo genome; 3) Screening all polymorphic sites in the male-specific region (NRY) of the ginkgo genome; 4) In the autosomal region, try to make the SNP sites evenly distributed in the whole ginkgo genome. After strict screening, 53,363 SNP sites that met the standards were obtained. According to the probe design principle, the 22bp sequence upstream and downstream of the site needs to be compared with any other region of the ginkgo genome, and sites with a matching degree of >85% are removed to ensure probe specificity; the probe length is 120bp, and the probe GC content is guaranteed to be 40%-60%, the annealing temperature is 55-65℃, the number of consecutive bases does not exceed 5, and the number of probe variable sites does not exceed 3. Probes were designed for sites that met the above design principles (two forward and reverse probes for each SNP site), and 45,776 pairs of capture probes were finally synthesized as probe combinations. The above probe combinations and targeted capture sequencing reagents were further used to obtain a high-density 45K SNP liquid phase chip for the whole genome of Ginkgo. The locations of the SNP sites included in the liquid phase chip on the chromosome are shown in Table 1, and their distribution on the chromosome is shown in Figure 1 , the distance between each SNP site is greater than 200bp.

[0158] Example 2 Performance evaluation of the Ginkgo whole genome 45K SNP liquid phase chip

[0159] (1) 10 samples of ginkgo were collected from Tianmu Mountain, Lin'an District, Hangzhou City, Zhejiang Province as validation samples, and genotyping was performed using the Ginkgo Whole Genome 45K SNP Liquid Phase Chip in Example 1. After sequencing and data analysis, the average site capture rate of the chip was 94.73%. The statistical results of the site capture rates of each sample detected using the Ginkgo Whole Genome 45K SNP Liquid Phase Chip provided by the present invention are shown in Figure 2 .

[0160] (2) Five Ginkgo samples were randomly selected, each divided into six parts, and libraries were constructed for each part. The whole genome 45K SNP liquid phase chip of Ginkgo in Example 1 was used for genotyping detection (the steps were the same as in Example 1). After sequencing and data analysis, the average proportion of technical replicate samples with successful capture and consistent genotypes was 93.11%. The proportion of sites with consistent results in six technical replicates for each sample is shown in Figure 3 .

[0161] The test results of capture rate, typing accuracy, repeatability and other performances showed that the chip had high genotype detection rate and accuracy, good stability and excellent overall performance.

[0162] The above-described embodiments provide a detailed description of the technical solutions of the present invention. It should be understood that the above descriptions are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A Ginkgo whole genome SNP site combination, characterized in that: The Ginkgo whole genome SNP site combination includes 45776 SNP sites, the physical positions of the 45776 SNP sites are determined based on the male_Ginkgo_v3 reference genome alignment, and the information of the 45776 SNP sites is shown in Table 1.

2. A probe combination for detecting the SNP site combination of the whole genome of Ginkgo biloba according to claim 1.

3. Use of the Ginkgo whole genome SNP site combination according to claim 1 in preparing a Ginkgo whole genome liquid phase chip.

4. A Ginkgo whole genome liquid phase chip, characterized in that: Comprising the probe combination according to claim 2.

5. The Ginkgo whole genome liquid phase chip according to claim 4, characterized in that: The probe combination is used to identify the genotype of each SNP site in the Ginkgo whole genome SNP site combination.

6. The Ginkgo whole genome liquid phase chip according to claim 4, characterized in that: The liquid phase chip is a Ginkgo 45K liquid phase chip based on targeted capture sequencing.

7. Application of the Ginkgo whole genome SNP site combination according to claim 1, the probe combination according to claim 2 or the Ginkgo whole genome liquid phase chip according to any one of claims 4-6 in genetic diversity analysis, sex identification, geographical traceability analysis, kinship analysis, genotyping, gene mapping or molecular assisted breeding of Ginkgo germplasm resources.

8. A method for whole genome genotyping of Ginkgo biloba, characterized in that: The following steps are involved: S1. Obtain genomic DNA of the ginkgo sample to be tested; S2. Detecting the genomic DNA using the Ginkgo whole genome liquid phase chip according to any one of claims 4 to 6 to obtain raw data; S3. Analyze the raw data to obtain genotyping results.

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