Application of a set of 40K SNP loci in the whole genome of cabbage crops

Abstract

The present invention discloses an application of a group of 40K SNP sites in the whole genome of cabbage crops. Based on the genome resequencing data of 102 cabbage materials and HDEM reference genome information, the present invention obtains the SNP variation file of each material, further preliminarily screens the site information in the data, and finally selects 62533 sites, and further uses 12 cabbage species test samples to test the 62533 probes synthesized for the first time, and selects sites according to the test results, preferentially selects target segments with good uniformity and polymorphism, and ensures the overall uniform distribution on the chromosome, and finally selects 40399 target segments for liquid chip preparation. The present invention is suitable for genotype detection of cabbage crops, and can be used to develop 40K liquid chips for cabbage crops, and combined with targeted sequencing technology, the genotype of cabbage crops can be measured with high throughput and low cost.

Description

Technical Field

[0001] The invention relates to SNP sites of cabbage crops, and in particular to the application of a group of 40K SNP sites in the whole genome of cabbage crops. Background Art

[0002] Cabbage crops are a general term for the Brassica species of the genus Brassica in the family Cruciferae, including common vegetables such as broccoli, cauliflower, cabbage, as well as variants such as kale, Brussels sprouts, kale and kohlrabi. Cabbage crops have extremely rich variation types, but the chromosome sets are all CC (2n=18).

[0003] The effective use of molecular markers is an important means of functional genomics research in cabbage crops. With the development of second-generation sequencing technology, single nucleotide polymorphism (SNP) markers have been widely used because of their large number and wide distribution on chromosomes. At present, the genotype detection of SNP markers in cabbage crops is mainly based on genome resequencing or simplified genome sequencing, which is not only costly but also not conducive to the integration of data obtained in different batches. Solid-state chips can integrate genotype data obtained at different times and locations, but there is no solid-state chip developed for the CC genome. Although some researchers have used the 60K SNP chip of Brassica napus (chromosome group AACC, 2n = 38) developed by Illumina in the United States to genotype cabbage species, there are differences in the CC genome between the two species, and a large number of markers in the AA genome of the 60K chip cannot be effectively used in cabbage species, which increases the detection cost.

[0004] The working principle of targeted sequencing technology is to perform fixed-point capture based on the complementary binding of the target probe and the target sequence. First, the gDNA library is constructed for the material to be tested. At the same time, according to the principle of DNA complementarity, a probe covering the target SNP is designed at each site to be tested, and the target probe is labeled with biotin. Then, the biotin-labeled target probe is hybridized with the target region of the genome in a liquid state to form a double strand. Subsequently, the target probe carrying the biotin label is molecularly adsorbed using streptavidin-coated magnetic beads to capture the target hybridized with the probe. Finally, the captured target sequence is eluted, the target amplified, the library is constructed and sequenced, and the genotype of the target SNP is finally obtained.

[0005] Targeted sequencing technology has many advantages in genotyping detection: (1) Platform adaptability. No specific expensive equipment is required; (2) Marker flexibility. There is no limit on the starting sample amount and the number of marker sites. Sequencing and marker genotyping can be completed in the same tube, and new primers can be added to the system at any time without adjusting the existing primers. More importantly, based on the same set of high-density markers, different numbers of markers can be obtained by adjusting the sequencing depth; (3) Detection efficiency. Thousands of highly uniform PCR reactions can be achieved in a single PCR reaction system; (4) Information additivity. The results are highly reliable, and the repeatability of the same batch of libraries and simultaneous sequencing, the same batch of libraries and different time of machine loading, and different batches of libraries and simultaneous sequencing are all above 99.9%; (5) Broad spectrum of applications. It can be used to detect all SNP marker sites in animals, plants, and microorganisms, and has a wide range of uses in marker-assisted major gene selection, backcross breeding and background selection, multi-gene aggregation breeding, seed purity detection, and transgenic component identification. Summary of the invention

[0006] The purpose of the present invention is to provide an application of a group of 40K SNP sites in the whole genome of cabbage crops, which is suitable for cabbage crop genotype detection and can be used to develop a 40K liquid phase chip for cabbage crops, combined with targeted sequencing technology, to measure the genotype of cabbage crops with high throughput and low cost.

[0007] In order to achieve the above object, the present invention provides an application of a group of 40K SNP sites in the whole genome of cabbage crops, and the SNP site information is as follows:

[0008] Table 1

[0009]

[0010]

[0011]

[0012]

[0013]

[0014]

[0015]

[0016]

[0017]

[0018]

[0019]

[0020]

[0021]

[0022]

[0023]

[0024]

[0025]

[0026]

[0027]

[0028]

[0029]

[0030]

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038]

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057] Among them, C1 to C8 on the left side of the site number represent the chromosome where the site is located, the middle value represents the physical position of the site on the chromosome, and the right side represents the SNP base genotype of the site in the reference genome HDEM, and the reference genome is HDEM.

[0058] The application is selected from any one of the following: (1) application in cabbage species genotyping; (2) application in the preparation of cabbage crop gene chips; (3) application in the construction of cabbage crop genetic maps; (4) application in the analysis of kinship of cabbage crop materials; (5) application in the authenticity identification of cabbage crop material varieties; or (6) application in the QTL positioning of cabbage crops.

[0059] Preferably, the gene chip adopts a liquid phase chip, and based on the liquid phase probe capture technology, a set of chips containing 40K site information that can be used to detect the background sites of cabbage crops is designed and synthesized. The liquid phase chip adopts the GenoBaits technology system, and the working principle is that the probe designed based on the target SNP marker is complementary to the DNA of the test sample for targeted sequence binding and sequencing, so as to achieve the purpose of detecting the genotype of the target SNP site of the test sample.

[0060] The application of the cabbage crop genome-wide 40K SNP loci of the present invention has the following advantages:

[0061] The loci of the present invention can be used to develop a 40K liquid phase chip for cabbage crops. Combined with targeted sequencing technology, the genotype of cabbage crops can be measured with high throughput and low cost, which can greatly improve the accuracy of genotype detection, improve the detection level, reduce the detection cost, and provide important technical support for molecular breeding of cabbage crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 This is the result of the population evolution tree analysis based on all sites in the target segment of the present invention.

[0063] Figure 2 This is a high-density genetic map constructed based on the 40K SNP liquid phase chip of cabbage crops in the present invention.

[0064] Figure 3 This is the QTL positioning result of the cabbage crop petal color of the present invention. DETAILED DESCRIPTION

[0065] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0066] Example 1 Preparation of gene chip using 40K SNP loci in cabbage crops

[0067] The preparation of 40K gene chip of cabbage crops is as follows: through library construction, hybridization capture, library quality inspection and sequencing analysis, the 40K target site is finally determined.

[0068] 1. Construction of libraries of 12 representative cabbage crop materials (see Table 2)

[0069] Table 2 Cabbage species used for SNP site capture

[0070]

[0071] Add 100ng cabbage DNA, 4μL GenoBaits EndRepairBuffer, 3.1μL GenoBaits Endrepair Enzyme (Shijiazhuang Boredi Company) to a 0.2μL PCR tube, and make up to 20μL with water; place the reaction tube in a PCR instrument, 37℃20min, 72℃20min. Then add 2μL GenoBaits Ultra DNAligase, 8μL GenoBaitsUltra DNALigase Buffer, 2μL GenoBaitsAdapter (Shijiazhuang Boredi Company), 8μL Nuclease-free water, and place in a PCR instrument at 22℃ for 60min. Add 1.2 times GenoPrep DNAClean Beads (Shijiazhuang Boredi Company), mix well, place on a magnetic stand, let stand for 3min, and remove the supernatant. Add 100μL freshly prepared 80vol% ethanol. Incubate at room temperature for 30 seconds and remove the supernatant. Air-dry until all ethanol evaporates, add 10μL of GenoBaits PCRMaster Mix, 1μL of GenoBaits Uni_oligo, 5μL of GenoBaits DNA index (Shijiazhuang Boredy Company), and 4μL of Nuclease-free water. Resuspend the magnetic beads and place in a PCR instrument at 98℃ for 2min; 98℃ for 30s, 65℃ for 30s, and 72℃ for 40s for 6-8 cycles. 72℃ for 4min.

[0072] Then add 20μL GenoPrep DNAClean Beads, mix thoroughly and let stand for 5 minutes, remove the supernatant, add 100μL freshly prepared 80vol% ethanol. Incubate at room temperature for 30 seconds, remove the supernatant. Add 30μL Tris-HCl to resuspend the library, mix thoroughly, place on a magnetic stand, let stand for 3 minutes, and remove the supernatant solution to obtain the DNA library.

[0073] 2. Library hybridization capture

[0074] The purified DNA libraries of 12 cabbage materials were mixed in equal amounts and hybridized at 65°C for 2 hours. The hybridized libraries were eluted using different wash buffers. The eluted target libraries were further enriched to obtain a sufficient amount of libraries for the machine; the enriched libraries were purified to obtain the final libraries.

[0075] 3. Library quality inspection and sequencing

[0076] The final library DNA concentration was measured using a Qubit fluorescence quantifier, and agarose gel electrophoresis was used to detect whether the fragment size of the library DNA was between 350 and 400 bp. The constructed library was sequenced using the BGI MGISEQ2000 sequencer.

[0077] 4. Test data analysis

[0078] 1) Preliminary screening of site information

[0079] Based on the genome resequencing data of 102 cabbage materials (see Table 2) and the HDEM reference genome information, the SNP variation file of each material was obtained, and the site information in the data was further preliminarily screened. The screening criteria were:

[0080] (1) Screening sites with a minimum allele frequency (MAF) > 0.35, a SNP site detection rate > 90%, and a heterozygous rate < 10% as all candidate sites;

[0081] (2) If condition 1 is met, the gene coding region is given priority, and the preferred order is: terminator mutation > non-synonymous mutation > promoter mutation > synonymous mutation > 5'UTR and 3'UTR > intron mutation > intergenic region mutation;

[0082] (3) The loci are evenly distributed on the chromosome;

[0083] (4) To ensure uniform distribution of loci, MAF was relaxed to 0.05, heterozygosity rate to 50%, and genotype missing rate to 50%, and finally 62,533 loci were selected.

[0084] 2) Probe design and synthesis

[0085] Use GenoBaits probe design software to design probes for target sites:

[0086] (1) Probe length 110 bp, probe GC content between 30% and 70%, and number of homology regions ≤ 5;

[0087] (2) The selected region does not contain SSR and N regions to the greatest extent possible;

[0088] Single-stranded nucleotide synthesis was performed according to the nucleotide sequence designed above, and a DNA nucleotide sequence with a length of 110 bp and a biotin group modified at the 5' end was synthesized.

[0089] 3) Further screening of sites

[0090] The 62,533 probes synthesized for the first time were tested using 12 cabbage species test samples. Site selection was performed based on the test results, with priority given to target segments with good uniformity and polymorphism, and overall uniform distribution on the chromosomes was ensured. Finally, 40,399 target segments were selected for liquid phase chip preparation.

[0091] like Figure 2 As shown in Table 1, the distribution of 40K SNP sites on chromosomes. As shown in Table 1, the numbers of 40K SNP sites are shown. The left side of the site number C1 to C8 indicates the chromosome where the site is located, the middle value indicates the physical position of the site on the chromosome, and the right side indicates the SNP base genotype of the site in the reference genome HDEM (https: / / www.genoscope.cns.fr / projet_BKL / cgi-bin / gbrowse / boleracea / ).

[0092] The 40K SNPs of cabbage crops provided by the present invention can be used to prepare gene chips for genotyping of cabbage species. The application of these sites can greatly improve the accuracy of genotype detection, improve the detection level, reduce the detection cost, and provide important technical support for molecular breeding of cabbage crops.

[0093] Example 2 Application of 40K SNP Liquid Phase Chip in Genotyping of Cabbage Crops

[0094] The present invention selected 12 cabbage germplasms including broccoli, cauliflower, kale, and wild type (see Table 2), and used the cabbage crop 40K SNP liquid phase chip prepared in Example 1 to detect the genotypes of the 40KSNP in these 12 materials in Example 1. The detection rate of the 12 materials was 94.51-98.80%. By comparing the reference genome HDEM with the chip site, the accuracy of the detected site was 99.3%. The entire experimental process was completed based on the GenoBaits technology system. The specific experimental process is as follows:

[0095] 1. Extract genomic DNA from the sample to be tested and construct a sample library

[0096] DNA was extracted using the CTAB method, and the quality and concentration of the DNA were tested. The construction of the library mainly includes the following four steps: (1) using ultrasonic treatment to randomly physically break the DNA fragments; (2) using end repair reagents to repair the end fragment DNA and add A-tail; (3) using T4 ligase to connect the Y-type adapter to the DNA fragments; (4) using barcode primers and high-fidelity DNA polymerase to amplify the connected DNA, and using different barcode primers to label the samples.

[0097] 2. Use liquid phase gene chip to determine the genotype of the target sample at 40K SNP sites

[0098] Probe hybridization includes the following six steps: (1) Library mixing. Mix the DNA library, cabbage-specific blocker and universal adapter blocker in a tube and vacuum aspirate them into dry powder; (2) Library hybridization. Place the probe and hybridization buffer in a centrifuge tube and hybridize at 65°C for 16 hours on a PCR instrument; (3) DNA capture. Add 100 μL GenoBaits DNA Probe Beads to the reaction system completed in the previous step of hybridization, incubate at 65°C for 45 minutes on a PCR instrument to allow the magnetic beads to bind to the probe. Selectively select the target fragment and remove the non-target fragment; (4) Library amplification. Amplify the selected fragment using library amplification primers and high-fidelity DNA polymerase; (5) Product purification. Use Beckman AMPure Beads for two rounds of purification; (6) Library quality inspection. Use Qubit fluorescence quantification instrument to measure the DNA concentration of the library, and then use agarose gel electrophoresis to detect whether the fragment size of the library DNA is between 300 and 400 bp. The constructed DNA library was sequenced using the BGI MGISEQ2000 sequencer.

[0099] 3. Genotype data analysis

[0100] The sequencing data were aligned to the HDEM reference genome, and SNP detection was performed on the sequencing data using GATK software to obtain genotype data of 12 cabbage species. The genotype data were analyzed, and the results are shown in Table 3.

[0101] Table 3 Genotyping results of 12 cabbage materials using 40K SNP liquid phase chip

[0102]

[0103] Example 3 Application of 40K SNP liquid phase chip in cabbage crop material kinship analysis

[0104] The present invention uses 40K SNP liquid phase chip to detect the genotypes of 12 cabbage species including broccoli, cauliflower, pagoda cabbage, etc. (see Table 3), and constructs a phylogenetic evolutionary tree based on the detection results (see Figure 1 ), clarified the relationship between these materials, and provided valuable reference for hybridization and molecular breeding between materials.

[0105] Example 4 Application of 40K SNP Liquid Phase Chip in Genetic Map Construction of Cabbage Crops

[0106] A BJ-F2 population of 214 plants was constructed by hybridizing broccoli (yellow petals) and kale (white petals). The DNA of each plant seedling leaf was extracted using the method mentioned in Example 2, and the population was genotyped using the 40K SNP liquid phase chip of the cabbage crop. Finally, a high-density genetic linkage map containing 4425 bins was constructed, with an average marker density of 2.13 cM / bin (see Figure 2 ).

[0107] Example 5 Application of 40K SNP Liquid Phase Chip in QTL Mapping of Cabbage Crops

[0108] Through the investigation of petal phenotype data of the population, the major QTL controlling the petal color of cabbage crops was located on chromosome C3 at 1077.8-1154.1 cM (see Figure 3 , the QTL positioning result is shown above, and the additive effect value is shown below). The physical position of the QTL mapped to the HDEM reference genome is 61.572-64.134 Mb. Candidate gene prediction analysis found that gene BolC3t20458H is likely to be the key gene regulating the petal color of cabbage crops, and its Arabidopsis homologous gene is Carotenoid cleavage dioxygenase 4 (CCD4). This is consistent with the results of the previously published article Map-based cloning and characterization of BoCCD4, a gene responsible for white / yellow petal color in B.oleracea, BMC Genomics, (2019) 20:242, indicating the accuracy of the liquid phase chip in genetic map construction and QTL positioning of important agronomic traits.

[0109] The application of the loci of the present invention can greatly improve the accuracy of genotype detection of cabbage crops, improve detection efficiency, reduce detection costs, and provide important technical support for molecular breeding of cabbage crops.

[0110] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. Application of a group of 40K SNP loci in the whole genome of cabbage crops, characterized in that: The SNP site information is as follows: Among them, the left side C1~C8 in the site number indicates the chromosome where the site is located, the middle value indicates the physical position of the site on the chromosome, and the right side indicates the SNP base genotype of the site in the reference genome HDEM, and the reference genome is HDEM; The application is selected from any one of the following: (1) Application in genotyping of cabbage species; (2) Application in the preparation of gene chips for cabbage crops; (3) Application in the construction of genetic maps of cabbage crops; (4) Application in the analysis of genetic relationships of cabbage crop materials; (5) Application in authenticity identification of cabbage crop varieties; or (6) Application in QTL localization in cabbage crops.

Images