A malus hallings 45k genome liquid chip and application

Abstract

The application relates to the technical field of whole genome gene chips, and specifically discloses a Malus hallings 45K genome liquid chip and application. The liquid chip genotyping site comprises 45217 SNP sites taking a Malus hallings reference genome as a reference. The Malus hallings 45K genome liquid chip can be used for Malus hallings evolution analysis, Malus hallings genetic breeding and Malus hallings genotyping, and the SNP sites of the chip uniformly cover the genome and are rich in functional sites, so that an accurate, reliable and standardized genotyping platform is provided for large-scale genotyping of a training population for Malus hallings genome selection research, the development of molecular marker assisted breeding and genome selection breeding is promoted, the breeding cycle of Malus hallings can be shortened, and selection precision and breeding efficiency can be improved.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of whole genome gene chips, and relates to a Catalpa bungeana 45K genome liquid chip and application. BACKGROUND

[0002] Catalpa bungeana is a broad-leaved tree of Catalpa in Bignoniaceae, is a famous precious timber tree species and an ornamental tree species in China, has good timber properties, has the effect of preventing wind and fixing sand, integrates the advantages of wood, ornamental and environmental protection, is widely welcomed by the public, is a precious timber tree species in China, and is mainly distributed in Henan, Gansu and Shandong, and the northernmost distribution is in Beijing. Catalpa bungeana has a long history. According to research, Catalpa bungeana has been distributed in North China and other regions since the Eocene before the Quaternary glacial period, and is one of the few ancient living fossil tree species that have been preserved after the structural changes of the prehistoric geology and geomorphology in China. Based on the above characteristics of Catalpa bungeana such as ornamental, practical, environmental protection and historical and cultural, it is necessary to study the genetic diversity, gene positioning and analysis and molecular marker screening of Catalpa bungeana.

[0003] The liquid chip is a new SNP detection method based on targeted sequencing, and the working principle is that the target probe is complementary combined with the target sequence for site capture and sequencing. The liquid chip detection has the characteristics of high throughput, high sensitivity and parallel detection; at present, the liquid chip has been applied to whole genome SNP determination of corn, Chinese cabbage, spruce, citrus, pineapple, melon and pepper, and has also been applied to whole genome SNP determination of cattle, sheep, pig, chicken and fish, and a small amount of research is about whole genome SNP determination of viruses, anaerobes and bacteria.

[0004] However, so far, there is no genome liquid chip for Catalpa bungeana. Therefore, developing a genome liquid chip for Catalpa bungeana has important significance for studying the genetic diversity analysis, gene positioning and cloning and molecular marker assisted screening of Catalpa bungeana. SUMMARY

[0005] In order to facilitate the genetic diversity analysis, gene positioning and cloning and molecular marker assisted screening of Catalpa bungeana, the application provides a Catalpa bungeana 45K genome liquid chip and application.

[0006] In a first aspect, the application provides a Catalpa bungeana 45K genome liquid chip, which adopts the following technical scheme:

[0007] A Catalpa bungeana 45K genome liquid chip, wherein the liquid chip genotyping site comprises 45217 SNP sites taking the Catalpa bungeana reference genome as a reference.

[0008] Optionally, the Schisandra 45K genome liquid chip further comprises functional sites, and the functional sites comprise 705 SNP sites and 35 InDel sites.

[0009] Optionally, the screening principles of the liquid chip genotyping sites comprise:

[0010] The probe length is 100-120 nt; the probe GC content is in the range of 30-70%; and the homologous region fragment is ≤5.

[0011] Further optionally, the screening principles of the liquid chip genotyping sites further comprise: uniform distribution of each site on the chromosome, site deletion rate <15%, site heterozygosity <50%, and MAF >0.35, and MAF >0.1 in a large GAP region.

[0012] Optionally, the liquid chip comprises 90734 mSNP sites with the Schisandra reference genome as the reference.

[0013] In a second aspect, the present application provides a use of the above-mentioned Schisandra 45K genome liquid chip in Schisandra evolution analysis.

[0014] In a third aspect, the present application provides a use of the above-mentioned Schisandra 45K genome liquid chip in Schisandra genetic breeding.

[0015] Optionally, the genetic breeding is selected from one or more of the following: germplasm resource genetic evaluation, population genetic structure analysis, kinship identification, quantitative trait genetic positioning, whole genome association analysis, and whole genome selection analysis.

[0016] In a fourth aspect, the present application provides a use of the above-mentioned Schisandra 45K genome liquid chip in Schisandra genotyping.

[0017] In a fifth aspect, the present application provides a use of the above-mentioned Schisandra 45K genome liquid chip in Schisandra resource protection.

[0018] In summary, the present application has the following beneficial effects:

[0019] The Schisandra 45K genome liquid chip of the present application comprises 45217 SNP sites, which uniformly covers the genome and is rich in functional sites, and can be used for Schisandra genotyping, evolution analysis, and genetic breeding, etc. It provides an accurate, reliable, and standardized genotyping platform for large-scale genotyping of training populations in Schisandra genome selection research, and helps to promote molecular marker-assisted breeding and genomic selection breeding, shorten the forest breeding cycle, and improve the selection accuracy and breeding efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1is the distribution of SNP sites in each chromosome in the 45K genome liquid chip of Malus halliana in Example 1 and resequencing data;

[0021] Figure 2 is the distribution map of SNP sites in each chromosome in the 45K genome liquid chip of Malus halliana in Example 1;

[0022] Figure 3 is the MAF distribution map of SNP sites in the 45K genome liquid chip of Malus halliana in Example 1;

[0023] Figure 4 is the gene structure annotation comparison map of randomly selected and replaced sites;

[0024] Figure 5 is the gene annotation distribution map of 45K sites after replacing sites;

[0025] Figure 6 is the PCA map of 375 materials of Malus halliana, Malus grijsii and Malus halliana based on the 45K genome liquid chip of Malus halliana in Example 1;

[0026] Figure 7 is the phylogenetic tree analysis map based on the 45K genome liquid chip of Malus halliana in Example 1;

[0027] Figure 8 is the phylogenetic tree analysis map of 300 Malus halliana natural populations based on the 45K genome liquid chip of Malus halliana in Example 1.

[0028] Figure 9 is the number distribution of each site of mSNP in each chromosome in Example 3. DETAILED DESCRIPTION

[0029] The present application is further described in detail below with reference to the accompanying drawings and examples, and it is particularly pointed out that the following examples are not to be construed as limiting the application. In the following examples, unless otherwise specified, the conventional conditions or the conditions recommended by the manufacturer are used, and the raw materials used in the following examples can be obtained from ordinary commercial sources unless otherwise specified.

[0030] The meaning of raw reads is original sequencing data; the meaning of clean reads is data filtered under certain conditions based on raw reads.

[0031] Example 1

[0032] Design and preparation of 45K genome liquid chip of Malus halliana

[0033] I. Population type and SNP genotyping

[0034] Sample source of Malus hallings: The development of 45K genome liquid chip of Malus hallings was based on 10X re-sequencing data of Malus hallings, Malus yunnanensis, Malus sieboldii and Malus prunifolia. Among them, the source of Malus hallings included 800 strains of Malus hallings and Malus prunifolia germplasm resource population, which were distributed in Beijing, Hebei, Henan, Shandong, Anhui and other places; and 75 strains of Malus sieboldii and Malus yunnanensis core population.

[0035] The specific steps are briefly described as follows:

[0036] 1. DNA extraction:

[0037] Healthy mature leaves were collected, naturally air-dried, and then punched into discs using a puncher and placed into a 96-well plate. The DNA was extracted using a magnetic bead method polysaccharide polyphenol plant tissue genomic DNA extraction kit.

[0038] 2. Library construction and determination:

[0039] The re-sequencing library was constructed using a GenoBaits DNA-seq Library Prep kit for DNA that passed quality inspection.

[0040] The re-sequencing library was preliminarily quantified using Qubit2.0, and the effective concentration of the re-sequencing library was accurately quantified using qPCR to ensure the quality of the library.

[0041] 3. Library sequencing:

[0042] After the library quality passed detection, sequencing was performed using a Huada MGI-2000 / MGI-T7 sequencing platform, and the sequencing mode was PE150 mode.

[0043] 4. Data processing:

[0044] The raw sequencing data was obtained by sequencing data quality control, reference genome alignment, variant detection and annotation (including SNP, InDel). The raw reads were filtered using software fastp (version 0.20.0, parameters: -n 10-q 20-u40) to obtain clean reads. The software BWA was used to align the quality-controlled clean reads with the reference genome sequence of Catalpa bungei (GSA number CRA016501, GSA submission number subCRA026045 published by the National Information Center; can be queried through https: / / ngdc.cncb.ac.cn / gsa / search?searchTerm=CRA016501) to locate the position of clean reads on the reference genome. According to the alignment results of clean reads on the reference genome, the HaplotypeCaller module of software GATK (version 4.2.1.0) was used for variant detection, and the detection parameters were: GATK Best Practices. The VariantFiltration module was used for filtering, and the SNP was annotated with the aforementioned uploaded database of Catalpa bungei reference genome sequence, and ANNOVAR [4] was used for gene function annotation.

[0045] II, site selection and design

[0046] The site selection method is:

[0047] 1. After merging the resequencing data of different batches from the above Catalpa bungei samples, the data was filtered according to the sequencing depth ≥ 5X; the deletion rate is less than 20%, and the MAF is greater than 0.1, to obtain 2,820,702 sites, and the average MAF = 0.123, see Table 1 for specific results.

[0048] Table 1 Catalpa bungei natural population 14M site data statistics

[0049]

[0050] 2. After site evaluation, the following three conditions were referred to continue to screen sites: ① probe length 100-120 nt; ② probe GC content in the range of 30-70%; ③ homologous region fragment ≤ 5; a total of 929,006 (accounting for 32.9%) sites were reserved.

[0051] 3. Follow the principle of uniform coverage of the genome, screen sites that meet the following conditions: ① NA < 15%; ② MAF > 0.35, MAF > 0.1 in large GAP region; ③ Het < 50%; and take the way of reducing the proportion of Intergenic sites and relaxing the parameters in the genomic Gap region, finally select 44483 sites of Catalpa liquid chip for constructing Catalpa liquid chip Catalpabungei 45K GBTS panel. The specific information of the obtained Catalpa liquid chip sites is shown in Table 2.

[0052] Table 2 Information of 44483 SNP sites of Catalpa liquid chip

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[0180] Comparison of resequencing data and chromosome distribution at 45K loci

[0181] Catalpa liquid phase chip The Catalpabungei 45K GBTS panel actually contains 45,217 core SNP loci, which are essentially 45K loci. Chromosomal distribution analysis was performed on the resequencing 2.8M loci and these 45K loci; the specific results are shown below. Figure 1 .

[0182] Figure 1 The results showed that in regions with dense resequencing data, 45K sites were also relatively dense; in regions with sparse resequencing blanks, 45K sites were preserved as much as possible to ensure full coverage of the genome. Figure 2 The results showed that the 45K site was evenly distributed on the chromosome.

[0183] Catalpa 45K liquid-phase chip site polymorphism and gene annotation

[0184] When screening for the 45K locus, the following conditions were met: 1. Initial MAF > 0.35; 2. Locus deletion rate < 15%; 3. Heterozygosity rate < 50%; 4. Even distribution on chromosomes; 5. Gap filling as much as possible, with the MAF relaxed to > 0.1; 6. Increasing the number of loci in exonic regions; etc. During development, the MAF was appropriately lowered, and the insufficient portion was supplemented with MAF > 0.2; if other conditions remained unchanged, selection was repeated; if the number of loci was still insufficient, loci were selected from regions with even smaller MAFs. Figure 3 As shown, the average minor allele frequency of the core SNP at the 45K locus is 0.33, indicating high polymorphism. Specifically, SNPs with MAF > 0.1 account for 99.31%, SNPs with MAF > 0.2 account for 89.50%, and SNPs with MAF > 0.3 account for 55.17%.

[0185] Under the premise of ensuring the uniform distribution of SNP sites, the intergenic SNP sites are replaced by the adjacent sites with the exonic region or with the biological information functional annotation. The adjacent sites refer to the adjacent sites. As shown in Figure 4 and Figure 5 Compared with the random sites, the number of functional regions (i.e. exonic region) of the 45K site data set after replacement is increased. The proportion of intergenic SNP sites is reduced by 1 / 5, the proportion of intergenic SNP sites is reduced from 47.81% to 39.49%, the proportion of exonic SNP sites is increased by 1 / 4, and the proportion of exonic site number is 29.20%.

[0186] Example 2

[0187] On the basis of the 45K genome liquid chip of Malus halliana obtained in Example 1, combined with the published academic papers, master and doctoral theses and the GWAS and QTL researches currently carried out by the research group, the sites significantly related to the wood properties, tree height, diameter at breast height and other properties of Malus halliana are collected, and then selected as the prospective markers to be added to the 45K chip. A total of 1188 SNP and indel sites are collected, and after probe evaluation, a total of 740 probes are designed successfully, with a success rate of 62.29%, and the specific results are shown in Table 3. The 740 probe design successful sites are called functional sites, including 705 SNP sites and 35 InDel sites, and the specific site information is shown in Table 4 and Table 5.

[0188] Table 3 Functional sites in Malus halliana chip

[0189]

[0190] Table 4 Information of 705 SNP functional sites

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[0195] Note: The bold site is the repeated site with the background site

[0196] Table 5 Information of 35 InDel functional sites

[0197]

[0198] Application example

[0199] Application example 1

[0200] Application of Catalpa bungei 45K genome liquid chip in evolution analysis of different tree species

[0201] As shown in Figure 6 and Figure 7 : Catalpa bungei 45K genome liquid chip of Example 1 can clearly distinguish different tree species, and the chip and resequencing results are similar. The 45K sites are representative, and can represent the analysis results of high-density SNP resequencing data with fewer sites.

[0202] Application Example 2

[0203] Application of Catalpa bungei 45K genome liquid chip in intra-species genetic evolution analysis of Catalpa bungei

[0204] Based on the Catalpa bungei 45K liquid chip data, genetic evolution analysis was performed on 300 materials of Catalpa bungei germplasm resource population, and the results are shown in Figure 8 : The population clustering is basically consistent with the source place, especially the resources from Beijing area are all clustered on one branch of the evolution tree; the 6 series numbers from Anhui are distributed on each branch; part of the Henan germplasm series numbers are relatively concentrated, and the other part is mixed with the series numbers from Hubei.

[0205] Based on the high-throughput resequencing data of Catalpa bungei natural population, Catalpa bungei var. pruinosa and Catalpa bungei var. pubescens core planting population, Catalpa bungei 45K genome liquid chip was selected, designed and developed. The SNP sites in the chip uniformly cover the genome, and the functional sites are rich, which provides an accurate, reliable and standardized typing platform for large-scale genotyping of training population of Catalpa bungei genome selection, helps to promote the development of molecular marker assisted breeding and genome selection breeding, can shorten the breeding cycle of Catalpa bungei, and improve the selection accuracy and breeding efficiency.

[0206] Example 3: Screening of mSNP sites of Catalpa bungei 45K genome liquid chip

[0207] Catalpa bungei 45K genome liquid chip includes 90734 mSNP sites in the detected segment, covering 45217 core SNP sites.

[0208] The molecular marker segment of the present application preferably includes 90734 mSNP (multi-nucleotide polymorphism) sites; the distribution of the 90734 mSNP sites on 20 chromosomes is as follows: Figure 96732, 5191, 8312, 8568, 4953, 3985, 4270, 7711, 4138, 1646, 6542, 3564, 4970, 5817, 2077, 2694, 5457, 1952, 1200, 955. The 45217 marker segments and the contained 90734 mSNP sites increase the number of sites, ensure the requirements of scientific research applications, and greatly reduce the use cost.

[0209] The embodiments are only an explanation of the present application, and are not a limitation of the present application. Those skilled in the art can make modifications to the embodiments without creative contribution after reading the specification, as long as the modifications are within the scope of the claims of the present application.

Claims

1. A Malus hupehensis 45K genome liquid chip, characterized in that, The liquid phase chip genotyping sites include 45217 sites with the reference genome of Cornus macrozona as the reference; The Cornus macrozona 45K genome liquid phase chip includes probes for detecting a combination of Cornus macrozona snp molecular markers, the combination of Cornus macrozona snp molecular markers is composed of 45217 snp sites, the specific positions of the snp sites are determined by comparing the sequence with the GSA number CRA016501 published by the National Information Center, and the GSA submission number is subCRA026045; the specific information of the snp sites is as follows:

2. The Illigens 45K genome liquid chip of claim 1, wherein, The Cornus macrozona 45K genome liquid phase chip further includes functional sites, the functional sites are composed of 705 SNP sites and 35 InDel sites, the information of the 705 SNP sites is as follows: The information of the 35 InDel sites is as follows:

3. The application of the Cornus macrozona 45K genome liquid phase chip in genetic breeding of Cornus macrozona according to any one of claims 1-2, the genetic breeding is selected from one or more of genetic evaluation of germplasm resources, population genetic structure analysis, kinship identification, quantitative trait genetic positioning, whole genome association analysis and whole genome selection analysis.

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