Catalpa bungei 45K genome liquid chip and application thereof

Abstract

The invention relates to the technical field of whole genome gene chips, and particularly discloses a catalpa bungei 45K genome liquid chip and application. The genetic typing sites of the liquid phase chip comprise 45217 SNP (Single Nucleotide Polymorphism) sites taking a catalpa bungei reference genome as a reference. The catalpa bungei 45K genome liquid chip can be used for catalpa bungei evolutionary analysis, catalpa bungei genetic breeding, catalpa bungei genetic typing and the like; the SNP sites of the chip uniformly cover a genome, the functional sites are rich, an accurate, reliable and standardized typing platform is provided for large-scale genotyping of a training group for catalpa bungei genome selection research, the development of molecular marker assisted breeding and genome selective breeding can be promoted, the catalpa bungei breeding period can be shortened, and the production cost is reduced. And the selection precision and the breeding efficiency are improved.

Description

Technical Field

[0001] The present application relates to the technical field of whole genome gene chips, and more particularly to a 45K genome liquid phase chip of Catalpa ovata and its application. Background Art

[0002] Catalpa ovata is a broad-leaved tree of the Bignoniaceae family. It is a famous precious timber species and garden ornamental tree species in my country. It not only has good wood properties, but also has the function of preventing wind and fixing sand. It combines the advantages of timber, ornamental and environmental protection. It is widely welcomed by the public and is a precious timber species in my country. It is mainly distributed in Henan, Gansu, Shandong and other places, and the northernmost is distributed in Beijing. Catalpa ovata has a long history. According to research, catalpa ovata has been distributed in North China and other regions as far back as the Eocene before the four-season glaciers in the geological era. It is one of the few ancient living fossil tree species that has been preserved after the changes in China's prehistoric geological and geomorphological structures. Based on the above-mentioned ornamental, practical, environmentally friendly and historical and cultural characteristics of catalpa ovata, it is necessary to conduct research on genetic diversity, gene positioning and analysis, and molecular marker screening of catalpa ovata.

[0003] Liquid phase chip is a new SNP detection method based on targeted sequencing. Its working principle is based on the complementary binding of target probes and target sequences for fixed-point capture and sequencing. Liquid phase chip detection has the characteristics of high throughput, high sensitivity and parallel detection. At present, liquid phase chip has been applied to the whole genome SNP determination of plants such as corn, cabbage, spruce, citrus, pineapple, melon and pepper. In addition, it has also been applied to the whole genome SNP determination of animals such as cattle, sheep, pigs, chickens and fish. There are also a small number of studies on the whole genome SNP determination of viruses, anaerobic bacteria and bacteria.

[0004] However, there is no liquid phase genome chip for Catalpa ovata so far. Therefore, developing a liquid phase genome chip for Catalpa ovata is of great significance for the study of genetic diversity analysis, gene localization and cloning, and molecular marker-assisted screening of Catalpa ovata. Summary of the invention

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

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

[0007] A 45K Catalpa ovata genome liquid phase chip, wherein the liquid phase chip genotyping sites include 45217 SNP sites with the Catalpa ovata reference genome as a reference.

[0008] Optionally, the Catalpa elata 45K genome liquid phase chip also includes functional sites, and the functional sites include 705 SNP sites and 35 InDel sites.

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

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

[0011] Further optionally, the screening principles of the liquid phase chip genotyping loci also include: each locus is evenly distributed on the chromosome, the locus deletion rate is less than 15%, the locus heterozygosity rate is less than 50%, and MAF is greater than 0.35, and MAF in large GAP regions is greater than 0.1.

[0012] Optionally, the liquid phase chip includes 90,734 mSNP sites based on the Catalpa ovata reference genome.

[0013] In a second aspect, the present application provides an application of the above-mentioned Catalpa ovata 45K genome liquid phase chip in the evolutionary analysis of Catalpa ovata.

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

[0015] Optionally, 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.

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

[0017] In a fifth aspect, the present application provides an application of the above-mentioned Catalpa ovata 45K genome liquid phase chip in the protection of Catalpa ovata resources.

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

[0019] The 45K liquid phase chip of the catalpa elata genome of the present application contains 45,217 SNP sites. The 45K liquid phase chip of the catalpa elata genome evenly covers the genome and is rich in functional sites. It can be used for various studies such as genotyping, evolutionary analysis, and genetic breeding of catalpa elata. It provides an accurate, reliable, and standardized typing platform for large-scale genotyping of training populations in catalpa elata genome selection research, which helps promote molecular marker-assisted breeding and genomic selection breeding, shorten the forest breeding cycle, and improve selection accuracy and breeding efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1The comparison of the distribution of SNP sites on each chromosome between the resequencing data and the liquid phase chip of the Catalpa ovata 45K genome in Example 1;

[0021] Figure 2 This is a distribution diagram of SNP sites on each chromosome in the Catalpa ovata 45K genome liquid phase chip of Example 1;

[0022] Figure 3 is the MAF distribution diagram of the SNP sites in the Catalpa ovata 45K genome liquid phase chip of Example 1;

[0023] Figure 4 It is a comparison diagram of gene structure annotations of randomly selected and replaced sites;

[0024] Figure 5 This is the 45K locus gene annotation distribution map after the site is replaced;

[0025] Figure 6 This is a PCA diagram of 375 materials of Catalpa yunnanensis, Catalpa truncatula and Catalpa ovata based on the Catalpa truncatula 45K genome liquid phase chip of Example 1;

[0026] Figure 7 This is a phylogenetic tree analysis diagram based on the Catalpa ovata 45K genome liquid phase chip of Example 1;

[0027] Figure 8 This is an evolutionary tree analysis diagram of 300 natural populations of Catalpa ovata based on the Catalpa ovata 45K genome liquid phase chip of Example 1.

[0028] Fig. 9 This is the quantitative distribution of each mSNP site in Example 3 on the chromosome. DETAILED DESCRIPTION

[0029] The present application is further described in detail below in conjunction with the accompanying drawings and examples. It is particularly noted that if no specific conditions are specified in the following examples, the experiments are carried out under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the raw materials used in the following examples can all be sourced from common commercial sources.

[0030] Raw reads refer to original sequencing data; clean reads refer to data filtered under certain conditions based on raw reads.

[0031] Example 1

[0032] Design and preparation of 45K liquid phase microarray of Catalpa ovata genome

[0033] I. Population type and SNP genotyping

[0034] Source of Catalpa samples: The development of Catalpa 45K genome liquid phase chip is based on 10× resequencing data of Catalpa ovata, Catalpa yunnanensis, Catalpa truncatum and Catalpa chinensis. Among them, the source of Catalpa ovata includes 800 lines of Catalpa ovata and Catalpa chinensis germplasm resource populations, and the planting areas of Catalpa ovata are distributed in Beijing, Hebei, Henan, Shandong, Anhui and other places; it also includes 75 lines of Catalpa truncatum and Catalpa yunnanensis core populations.

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

[0036] 1. Extract DNA:

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

[0038] 2. Library construction and determination:

[0039] The GenoBaits DNA-seq Library Prep Kit was used to construct a resequencing library for DNA that passed the quality inspection.

[0040] Qubit2.0 was used to perform preliminary quantification of the resequencing library, and the effective concentration of the resequencing library was accurately quantified using the qPCR method to ensure the quality of the library.

[0041] 3. Library sequencing:

[0042] After the library quality test was qualified, sequencing was performed using the BGI-2000 / MGI-T7 sequencing platform in the PE150 mode.

[0043] 4. Data processing:

[0044] The original sequencing sequence was obtained after sequencing data quality control, reference genome alignment, variation detection and annotation (annotation content includes SNP, InDel). The raw reads were filtered using fastp (version 0.20.0, parameters: -n 10-q 20-u40) to obtain clean reads. The clean reads after quality control were aligned with the reference genome sequence of Catalpa ovata (the GSA number published by the National Information Center is CRA016501, and the GSA submission number is subCRA026045; it can be queried through https: / / ngdc.cncb.ac.cn / gsa / search?searchTerm=CRA016501) using the software BWA to locate the position of the clean reads on the reference genome. According to the alignment results of the clean reads on the reference genome, the HaplotypeCaller module of the software GATK (version 4.2.1.0) was used for variation detection, and the detection parameters were: GATKBest Practices. The VariantFiltration module was used for filtering, and the SNPs were annotated with the current reference genome sequence of Catalpa ovata (unpublished). [4] Perform 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 samples, the data were filtered according to the sequencing depth ≥5X; the missing rate was less than 20%, and the MAF was greater than 0.1, and 2,820,702 sites were obtained, with an average MAF of 0.123. The specific results are shown in Table 1.

[0048] Table 1 Statistics of 14M loci of natural population of Catalpa ovata

[0049]

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

[0051] 3. Following the principle of uniform coverage of the genome, we screened sites that met the following conditions: ①NA<15%; ②MAF>0.35, MAF>0.1 in large GAP regions; ③Het<50%; and adopted the method of reducing the proportion of intergenic sites and relaxing the parameters in the genomic Gap region. Finally, 44,483 sites of the catalpa sap phase chip were selected for the construction of the catalpa sap phase chip. Catalpabungei 45K GBTS panel. The specific information of the Catalpa sap phase array loci obtained is shown in Table 2.

[0052] Table 2 Information on 44483 SNP loci in the liquid phase microarray of Catalpa ovata

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

[0181] Catalpa ovata liquid phase chip Catalpabungei 45K GBTS panel actually contains 45217 core SNP sites, that is, 45K sites. The chromosome distribution of the resequenced 2.8M sites and the 45K sites was analyzed. The specific results are shown in Figure 1 .

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

[0183] 45K liquid phase microarray site polymorphism and gene annotation of Catalpa ovata

[0184] When screening the 45K loci, the following conditions must be met: 1. Initially set MAF>0.35; 2. Locus deletion rate <15%; 3. Heterozygosity rate <50%; 4. Evenly distributed chromosomes; 5. Fill in GAPs as much as possible, and relax the MAF of the loci to>0.1; 6. Increase the number of loci in the exonic region; etc. During development, the MAF should be appropriately lowered, and the insufficient part should be supplemented with MAF>0.2; when other conditions remain unchanged, select again; and when there is still an insufficient number of loci, select loci in the area with a smaller MAF. Figure 3 As shown, the average MAF of the minor allele frequency of the core SNP sites of the 45K site is 0.33, and the site polymorphism is high. Specifically, the number of SNP sites with MAF>0.1 accounts for 99.31%, the number of SNP sites with MAF>0.2 accounts for 89.50%, and the number of SNP sites with MAF>0.3 accounts for 55.17%.

[0185] Under the premise of ensuring uniform distribution of SNP sites, the intergenic region SNP sites are replaced with adjacent sites that are located in the exon region or have biological information function annotations. The adjacent sites refer to adjacent sites. Figure 4 and Figure 5 As shown in the figure, compared with random sites, the proportion of functional regions (i.e. exon regions) of the genes in the 45K site dataset after replacement increased. The proportion of SNP sites in the intergenic region decreased by 1 / 5, and the proportion of SNP sites in the intergenic region decreased from 47.81% to 39.49%. The proportion of SNP sites in the exonic region increased by 1 / 4, and the number of sites in the exonic region accounted for 29.20%.

[0186] Example 2

[0187] On the basis of the 45K genomic liquid phase chip of catalpa obtained in Example 1, combined with the published academic papers, master's and doctoral dissertations and the GWAS and QTL studies currently being carried out by this research group, loci significantly associated with wood properties, tree height, diameter at breast height and other traits of catalpa wood were collected, and added to the 45K chip as prospect markers after additional selection. A total of 1188 SNP and indel sites were collected. After probe evaluation, a total of 740 probes were successfully designed, with a success rate of 62.29%. The specific results are shown in Table 3. The 740 probes were successfully designed, which are called functional sites, including 705 SNP sites and 35 InDel sites. The specific site information is shown in Tables 4 and 5.

[0188] Table 3 Functional sites in the Catalpa ovata chip

[0189]

[0190] Table 4705 SNP functional site information

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[0195] Note: The bold sites are sites that are repeated with the background sites.

[0196] Table 535 InDel functional site information

[0197]

[0198] Application Examples

[0199] Application Example 1

[0200] Application of Catalpa ovata 45K genome liquid phase microarray in evolutionary analysis of different tree species

[0201] like Figure 6 as well as Figure 7 As shown: the 45K liquid phase chip of the catalpa ovata genome of Example 1 can clearly distinguish different tree species, the chip and resequencing results are similar, the 45K sites are highly representative, and the analysis results of high-density SNP resequencing data can be represented by fewer sites.

[0202] Application Example 2

[0203] Application of 45K genomic liquid phase microarray in genetic evolution analysis within catalpa ovata species

[0204] Based on the 45K liquid phase chip data of Catalpa ovata, 300 materials of Catalpa ovata germplasm resource population were analyzed for genetic evolution. The results are as follows Figure 8 As shown: the clustering of the groups is basically consistent with the place of origin, especially the resources from Beijing, which are all clustered on a large branch of the evolutionary tree; the 6 lines from Anhui are distributed on various branches; some of the Henan germplasm lines are relatively concentrated, while the other part is mixed with the Hubei lines.

[0205] Based on the high-throughput resequencing data of natural populations of Catalpa ovata, core planting populations of Catalpa ovata and Catalpa yunnanensis, the Catalpa ovata 45K genomic liquid phase chip was selected, designed and developed. The SNP sites in this chip evenly cover the genome and are rich in functional sites, providing an accurate, reliable and standardized typing platform for large-scale genotyping of training populations in Catalpa ovata genomic selection research, which will help promote the development of molecular marker-assisted breeding and genomic selection breeding, shorten the breeding cycle of Catalpa ovata, and improve selection accuracy and breeding efficiency.

[0206] Example 3 Screening of mSNP sites using Catalpa ovata 45K genome liquid phase chip

[0207] The 45K liquid phase array of the Catalpa ovata genome includes 90,734 mSNP sites within the detected segment, covering 45,217 core SNP sites.

[0208] The molecular marker segment of the present application preferably includes 90734 mSNP (Multiple Nucleotide Polymorphism) sites; the distribution of the 90734 mSNPs on 20 chromosomes is as follows: Fig. 9): 6732, 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 90734 mSNP sites included increase the number of sites, ensuring the requirements of scientific research applications while greatly reducing the cost of use.

[0209] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A 45K genome liquid phase chip of Catalpa ovata, characterized in that: The liquid phase chip genotyping sites include a total of 45,217 sites based on the Catalpa ovata reference genome.

2. The Catalpa elata 45K genome liquid phase chip according to claim 1, characterized in that: The Catalpa elata 45K genome liquid phase chip also includes functional sites, which include 705 SNP sites and 35 InDel sites.

3. The Catalpa elata 45K genome liquid phase chip according to claim 1, characterized in that: The screening principles of the liquid phase chip genotyping loci include: The probe length is 100-120 nt; the probe GC content is in the range of 30-70%; the homologous region fragments are ≤5.

4. The Catalpa elata 45K genome liquid phase chip according to claim 3, characterized in that: The screening principles of the liquid chip genotyping sites also include: each site is evenly distributed on the chromosome, the site deletion rate is less than 15%, the site heterozygosity rate is less than 50%, and MAF is greater than 0.35, and MAF in large GAP regions is greater than 0.

1.

5. The Catalpa elata 45K genome liquid phase chip according to claim 1, characterized in that: The liquid phase chip includes 90734 mSNP sites with reference to the Catalpa ovata reference genome.

6. Use of the Catalpa elata 45K genome liquid phase chip according to any one of claims 1 to 5 in the evolutionary analysis of Catalpa elata.

7. Use of the Catalpa elata 45K genome liquid phase chip described in any one of claims 1 to 5 in Catalpa elata genetic breeding.

8. The use of the 45K genome liquid phase chip of Catalpa ovata in genetic breeding of Catalpa ovata according to claim 7, characterized in that: The genetic breeding is selected from one or more of germplasm resource genetic evaluation, population genetic structure analysis, kinship identification, quantitative trait genetic positioning, whole genome association analysis and whole genome selection analysis.

9. Use of the Catalpa elata 45K genome liquid phase chip according to any one of claims 1 to 5 in Catalpa elata genotyping.

10. Use of the Catalpa elata 45K genome liquid phase chip described in any one of claims 1 to 5 in the protection of Catalpa elata resources.

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