CN117660687A

CN117660687A - High-oil peanut whole genome molecular marker combination, probe, gene chip and application

Info

Publication number: CN117660687A
Application number: CN202311709863.1A
Authority: CN
Inventors: 许彦芬; 蓝昊发; 刘基生; 张仁旭; 张子豪; 张丛
Original assignee: Shijiazhuang Breeding Biotechnology Co ltd
Current assignee: Shijiazhuang Breeding Biotechnology Co ltd
Priority date: 2023-12-13
Filing date: 2023-12-13
Publication date: 2024-03-08

Abstract

The invention relates to the technical field of molecular genetic breeding and genotype detection, and particularly discloses a high-oil peanut whole genome molecular marker combination, a probe, a gene chip and application, wherein the high-oil peanut whole genome molecular marker combination comprises 2507 high-oil gene CDS regions and/or 8003 background SNP loci; the physical locations of 2507 high oil gene CDS regions and 8003 background SNP sites were determined based on whole genome sequence alignment of the peanut reference genome; the version number of the whole genome sequence of the peanut reference genome is gca_004170445.1_asm417044v1;2507 high oil gene CDS regions are shown as m 0001-2507; 8003 background SNP sites are shown as s 0001-8003. The invention defines the molecular marker combination of the high-oil peanut, designs a set of probes and gene chips for detecting the molecular marker combination of the high-oil peanut, and can be used for Gao Youhua genotyping, high-oil peanut breeding, peanut oil content whole genome prediction, gao Youhua haplotype evaluation and identification and high-oil-content backcross selection.

Description

High-oil peanut whole genome molecular marker combination, probe, gene chip and application

Technical Field

The invention relates to the technical field of molecular genetic breeding and genotype detection, in particular to a high-oil peanut whole genome molecular marker combination, a probe, a gene chip and application.

Background

Peanut (Arachis hypogaea L.) is one of four large oil crops in China, the average oil content is about 51%, and excellent ultrahigh oil resources with partial oil content exceeding 60% are found in germplasm resources, so that the oil content of peanut varieties is greatly improved. Therefore, the improvement of the oil content of the peanuts becomes a primary aim of improving the peanut quality in China.

At present, in the genetic improvement process of peanut oil content, traditional breeding plays a certain role, but the progress is slow. Moreover, the oil content of peanut germplasm resources cannot be subjected to phenotype selection directly through a traditional breeding means, and a molecular marker assisted breeding technology is adopted, particularly with the rapid reduction of high-throughput sequencing cost, SNP markers serving as third-generation molecular markers are increasingly applied to the identification and evaluation of phenotypes in assisted breeding.

However, in recent years, the basic research related to peanut oil content is slow due to the lack of peanut reference genome, and along with the decoding of peanut genome, the molecular markers related to peanut high oil state are searched and developed into a liquid phase chip together with background SNP, so that the method has important significance for the prediction of peanut oil content, the evaluation and identification of high oil peanut and the application of high oil state due to backcross selection.

Disclosure of Invention

The invention aims to provide a high-oil peanut whole genome molecular marker combination, a probe, a gene chip and application thereof, which define the high-oil peanut molecular marker combination, and designs a set of probe and gene chip which can be used for detecting the high-oil peanut molecular marker combination, and can be used for Gao Youhua genotyping, high-oil peanut breeding, peanut oil content whole genome prediction, gao Youhua haplotype evaluation identification and high-oil gene backcross selection.

The invention is realized by the following technical scheme:

the high-oil peanut whole genome molecular marker combination comprises 2507 high-oil gene CDS regions and/or 8003 background SNP loci; the physical locations of 2507 high oil gene CDS regions and 8003 background SNP sites were determined based on whole genome sequence alignment of the peanut reference genome; the version number of the whole genome sequence of the peanut reference genome is gca_004170445.1_asm417044v1,

the download address of the version number is: https:// www.ncbi.nlm.nih.gov/data/genome/GCA_ 004170445.1/;

the CDS region of the high-oil gene is expressed in a mode of physical position of chromosome number_start site_physical position of termination site; 2507 high oil gene CDS regions are shown in m0001-2507 in Table 1;

wherein, the background SNP locus is expressed by adopting a chromosome number_physical position mode; 8003 background SNP sites are shown as s0001-8003 in Table 2.

TABLE 1

TABLE 2

A set of nucleotide probes for detecting the whole genome molecular marker combination of the peanut with high oil content.

The gene chip is a liquid phase chip and contains a set of nucleotide probes for detecting the high-oil peanut whole genome molecular marker combination.

Further, the nucleotide probes include a combination of nucleotide probes for detecting 2507 high oil gene CDS regions; and/or nucleotide probe combinations for detecting 8003 background SNP sites.

An application of the high-oil peanut whole genome molecular marker combination or the gene chip in Gao Youhua genotyping.

The high-oil peanut whole genome molecular marker combination or the application of the gene chip in Gao Youhua breeding.

The high-oil peanut whole genome molecular marker combination or the application of the gene chip in peanut oil content whole genome prediction.

The application of the high-oil peanut whole genome molecular marker combination or the gene chip in Gao Youhua haplotype evaluation and identification.

The application of the high-oil peanut whole genome molecular marker combination or the gene chip in high-oil-content gene backcross selection.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. the liquid phase chip developed by the invention not only can detect high-oil genes, but also can be used for foreground selection of high-oil backcross transformation, estimation and identification of high-oil peanut haplotype, whole genome prediction of peanut oil content, and simultaneously, 8K background marks uniformly distributed in the whole genome are introduced, so that the liquid phase chip can be used for background selection of backcross transformation and application scene diversification.

2. The liquid phase chip for breeding the high-oil peanut varieties takes 8K variation sites selected from the whole genome according to uniform distribution as background marks; for peanut groups with more individuals, genotyping can be performed without re-sequencing, the single-sample single-site detection cost is as low as 0.02 yuan, the detection cost is greatly reduced, and the method has the advantage of low cost.

3. The liquid phase chip for breeding the high-oil peanut varieties adopts a targeted sequencing genotype detection (GBTS) technology, and can customize and add new and personalized genes or background site marks according to specific requirements of customers; the method has the advantages of no requirement of initial detection of sample size and flexible sample feeding.

4. Compared with whole genome re-sequencing, simplified genome sequencing and the like, the chip has a short detection period; the data to be processed is relatively less, the period required by data analysis is also shorter, the result can be ensured to be obtained by researchers and breeding workers in a relatively shorter period, and the method has the advantage of short period.

5. The single-site average depth of the SNP liquid-phase chip is more than 100X, the sequencing depth is about 10 times of the resequencing data, the accuracy of the result is higher, and the advantage of the result is accurate.

Drawings

The accompanying drawings, which are included to provide a further understanding of embodiments of the invention and are incorporated in and constitute a part of this application, illustrate embodiments of the invention. In the drawings:

FIG. 1 is a distribution diagram of 8003 background SNP sites of the invention in a whole genome;

FIG. 2 is a histogram of MAF distribution at 8051 sites in example 6;

FIG. 3 is a chromosome profile of 8051 sites in example 6.

Detailed Description

For the purpose of making apparent the objects, technical solutions and advantages of the present invention, the present invention will be further described in detail with reference to the following examples and the accompanying drawings, wherein the exemplary embodiments of the present invention and the descriptions thereof are for illustrating the present invention only and are not to be construed as limiting the present invention.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, it will be apparent to one of ordinary skill in the art that: no such specific details are necessary to practice the invention. In other instances, well-known structures, materials, or methods have not been described in detail in order to avoid obscuring the present invention.

Example 1:

selection of CDS region and background SNP locus of peanut high oil gene:

the nucleotide sequences of the published oil genes in arabidopsis and rice were downloaded from NCBI and aligned to the first cultivated peanut (peanut) that the company participated in the decoding (sketch information (Chen et al, 2019), the 2507 genes with the longest alignment and highest identity were selected from the alignment as homologs of the peanut oil genes, the CDS region of these genes included 28907 exons in total, and the physical position information of the CDS region of the full-length 3.97mb and 2507 high-oil genes is shown in table 1.

Background SNP locus selection was performed from a resequencing variation set of 34 peanut varieties with extensive genetic variation according to the following principle: 1) Site deletion rate <20%; 2) These allele frequencies MAF >0.35; 3) The heterozygosity rate is <5%; 4) A total of 9004 SNP sites were selected uniformly distributed on the chromosome.

Example 2:

obtaining nucleotide probe combinations:

the GenoBaits Probe Designer software is used for carrying out site evaluation (namely probe design) on homologous genes of peanut grease and sites selected according to a uniform distribution principle, wherein the evaluation principle is as follows: 1) The length of the probe sequence is 100bp; 2) The GC content of the probe sequence is 30% -80%; 3) The number of homologous regions of the probe in the whole genome of the peanut is less than or equal to 5; 4) The probe region does not contain SSR and unknown base N; 5) A total of 70345 probes covering the CDS region of 2507 candidate genes were obtained for each site 2x probe design, the total length of the coverage region was 4.38mb, and 18008 probes were designed for 9004 SNP sites. The probes are used as liquid phase chip candidate probes for breeding high-oil peanut varieties for subsequent test and evaluation.

The 70345 probe combinations obtained above were adjusted with 2507 high oil gene CDS regions in table 1, and finally a nucleotide probe combination capable of capturing 2507 snp segments was obtained.

The 18008 probe combinations obtained above were adjusted with 8003 background SNP sites in table 2, and finally a nucleotide probe combination capable of capturing 8003 background SNP sites was obtained.

Example 3:

a gene chip which is a liquid phase chip comprising the nucleotide probe combination capable of capturing 2507 CDS regions of high oil genes and/or the nucleotide probe combination capable of capturing 8003 background SNP sites in example 2.

Example 4:

genotype detection of test samples and breeding populations:

the method for genotype detection of 2507 high oil gene CDS regions and 9004 SNP sites in example 1 using the liquid phase chip developed in example 3, comprises the steps of:

step 1, genomic DNA extraction

Tender peanut leaf tissues are selected, nucleic acid extraction is carried out by using a plant genome DNA extraction kit produced by Shijia Boruidi biotechnology limited company, and the extracted DNA concentration is accurately quantified by using Qubit.

Step 2, DNA fragmentation and end repair

PCR amplification system: the DNA dosage is 300ng; 2.6 mu L of end repair enzyme and 4 mu L of buffer solution; the system was replenished to 20. Mu.L with no nucleic acid.

PCR amplification procedure: 20min at 37 ℃; 20min at 72 ℃.

Step 3, introducing a connector connecting system by PCR in the system of the step 2;

PCR system: a linker of 4. Mu.L; 2. Mu.L of ligase; buffer 8. Mu.L; the non-nucleic acid water was made up to 20. Mu.L.

PCR amplification procedure: and 60min at 22 ℃.

Step 4, purifying the product of the connection system;

adding magnetic beads into the PCR product, and vibrating uniformly;

4.2 standing for 5min at room temperature, and centrifuging briefly;

4.3, placing the PCR tube on a magnetic rack until the solution is clear;

4.4 carefully remove supernatant, ensure that no magnetic beads are attracted.

4.5 keeping the PCR tube/plate in a magnetic rack, adding 100 mu L of 80% ethanol, and standing at room temperature for 30s;

4.6 remove supernatant, keep PCR tube/plate in magnetic rack, place at room temperature until ethanol is completely volatilized.

And 5, performing PCR amplification by taking peanut genome DNA as a template to obtain a DNA library.

PCR amplification system: adding 10 mu L of a mixture of the PCR enzyme and the buffer solution into the product of the step 4; sequencing 2 μl of the barcode; the system was replenished to 20. Mu.L with no nucleic acid.

PCR amplification procedure: 2min 1 cycle at 98 ℃; (98 ℃ C. 30s,65 ℃ C. 30s,72 ℃ C. 40 s) 5 cycles; and 4min1 cycle at 72 ℃.

And 6, purifying the obtained DNA library.

6.1, adding 20 mu l GenoPrep DNAClean Beads into the product obtained in the step 6, vibrating and uniformly mixing, wherein bubbles are not generated in the vibrating process;

6.2 standing for 5min, and centrifuging briefly;

6.3 placing the PCR tube/plate on a magnetic rack until the solution is clear, and standing at room temperature for at least 3min;

6.4 remove supernatant, ensure that no magnetic beads are attracted.

6.5 keep the PCR tube/plate in the magnetic rack and add 100. Mu.L 80% ethanol. The mixture was left at room temperature for 30 seconds.

6.6 remove supernatant, keep PCR tube/plate in magnetic rack, place at room temperature until ethanol volatilizes.

6.7 taking out the PCR tube or plate from the magnetic frame, adding 35 μL of effect Buffer, shaking, mixing, standing for 5min, and centrifuging briefly

6.8, placing the PCR tube on a magnetic rack until the solution is clear;

6.9 the supernatant was transferred to a new 0.2ml low adsorption PCR tube/plate.

And 7, mixing the purified DNA library in equal amounts.

Step 8, concentrating the mixed library, adding the concentrated library into a hybridization system, and performing hybridization capturing by using the prepared probe;

PCR amplification system: 1.5-2.5 μg of the library; 300ng of probe; the system was made up to 16. Mu.l without nucleic acid water.

PCR amplification procedure: 95℃for 10min (thermal cover temperature 105 ℃); 65℃for 2-4h (thermal cover temperature 75 ℃).

Step 9, eluting to remove unbound DNA

9.1 after the hybridization in step 9 was completed, the lid of the PCR instrument was opened, the lid of the PCR tube was opened, and 16. Mu.L of the hybridization trapping solution was transferred to the prepared magnetic beads.

9.2 vortex shaking for 10s, fully and uniformly mixing, and instantaneous centrifuging.

9.3 placing the PCR tube into a PCR instrument at 65℃for 45min and a hot cover temperature of 75 ℃.

9.4 shaking for 5s every 12min, and instantaneous centrifugation.

9.5 100. Mu.L of pre-warmed elution buffer at 65℃was added to each PCR tube.

9.6 briefly vortex for 5s and centrifuge for 5s.

9.7 the PCR tube was placed on a magnetic rack until the beads were completely separated from the solution.

9.8 remove the supernatant with a pipette, retain the beads, and place the PCR tube into a PCR instrument at 65 ℃.

9.9 add 150. Mu.L of GenoBaits 1X Stringent Wash Buffer preheated at 65℃and gently suck 10 times up and down, mix well the beads. After the last group of samples were mixed, they were placed on a PCR instrument for 2min.

9.10 the PCR tube was placed on a magnetic rack until the beads were completely separated from the solution and the supernatant was removed rapidly with a pipette.

9.11 transfer the PCR tube from the magnetic rack, add 150. Mu.L of room temperature elution buffer I, shake for 2min.

9.12 the PCR tube was placed on a magnetic rack until the beads were completely separated from the solution. The supernatant was removed with a pipette.

9.13 transfer the PCR tube from the magnetic rack, add 150. Mu.L of elution buffer II at room temperature and shake for 1min.

9.14 the PCR tube was placed on a magnetic rack until the beads were completely separated from the solution. The supernatant was removed with a pipette.

9.15. Mu.L of elution buffer III at room temperature was added and the mixture was shaken for 30s.

9.16 the PCR tube was placed on a magnetic rack until the beads were completely separated from the solution. The supernatant was removed with a pipette.

9.17 the tube containing the captured DNA was removed from the magnet rack and 20. Mu.L of nuclease free water was added. The magnetic beads are sucked and beaten for 10 times slowly by a pipette, so that the resuspension of all the magnetic beads is ensured.

And step 10, enriching the library.

PCR system: step 9, enriching DNA on the magnetic beads; 15. Mu.L of enzyme mixture; 1.2. Mu.L of primer; the system was replenished to 30. Mu.L without nucleic acid.

PCR amplification procedure: 45s 1 cycle at 98 ℃; (98 ℃ C. 15s,60 ℃ C. 30s,72 ℃ C. 30 s) 13 cycles; 72 ℃ for 1min.

Step 11, purifying the enriched product to finish the preparation of the sequencing library

11.1, placing the PCR tube on a magnetic rack until the solution is clear;

11.2 transferring the supernatant to a new PCR tube;

11.3 adding 45 μl of magnetic beads into each reaction, and shaking and mixing.

11.4 standing at room temperature for 5min, and centrifuging briefly;

11.5, placing the PCR tube on a magnetic rack until the solution is clear;

11.6 removing the supernatant with a pipette;

11.7 keep the PCR tube on a magnetic rack and add 100. Mu.L 80% ethanol. Incubating for 30 seconds at room temperature;

11.8 removing the supernatant with a pipette;

11.9, keeping the PCR tube on a magnetic frame, and standing at room temperature until ethanol volatilizes;

11.10 the PCR tube was moved from the magnet holder to room temperature, 35. Mu.L of elution buffer was added, and mixed by shaking. After standing at room temperature for 5min, the mixture was centrifuged briefly.

11.11 placing the PCR tube on a magnetic rack until the solution is clear;

11.12 the supernatant was transferred to a new 0.2mL low adsorption tube, ensuring that no magnetic beads were attracted. The purified DNA library may be stored at-20℃until sequencing is desired.

Step 12, carrying out high-throughput sequencing on the equivalent mixed sequencing library by using a Huada T7 sequencer;

step 13, splitting data of the original sequencing bases according to the barcode of different materials, filtering low-quality sequencing data, comparing the sequencing data with a reference genome of the Hui peanut, and performing mutation mining;

and 14, obtaining corresponding genotypes according to the obtained SNP information.

Example 5:

optimization of liquid phase chip and whole genome molecular marker combination

In the embodiment, 6 peanut materials with excellent agronomic characters and wide genetic background differences are selected, and the liquid phase chip is subjected to preliminary test by adopting the method described in the embodiment 4. The results are shown in Table 3; the target rate in the whole liquid chip was 76.64%, the capture uniformity of 2507 high oil-based gene CDS region was 97.07%, the capture uniformity of 9004 background SNP sites was 93.17%, and 472 sites out of 9004 sites could not be detected under 2.5G sequencing.

Table 3: summary of test information of liquid phase chip in 6 parts of materials

Because the uniformity of the gene CDS region is obviously higher than that of SNP loci, in order to better save sequencing data volume and improve the cost performance of the liquid phase chip detection, 8003 loci which are better in uniformity and uniformly distributed are screened out from 9004 SNP loci and serve as final background SNP loci, probes in the liquid phase chip are correspondingly deleted, and chromosome distribution diagrams of the 8003 background loci are shown in fig. 1 and table 4; finally, a whole genome molecular marker combination, a probe and a liquid phase chip are obtained, wherein the whole genome molecular marker combination comprises 2507 high-oil gene CDS regions shown in table 1 and 8003 background SNP sites shown in table 2.

Table 4: liquid phase chip background site for peanut high-oil breeding is distributed in number of each chromosome

Example 6:

high-oil peanut whole genome molecular marker combination or application of the gene chip:

genotyping was performed on 244 peanut natural populations using the liquid phase chip optimized in example 5, the sequencing result was taken at 5x or more depth of sequencing as the threshold for region coverage and SNP identification, 2507 genes were 3.97Mb regions, 244 samples were covered 3.78Mb on average, the average coverage was 95.25%, the average detection rate of 8003 SNP sites was 95.25%, and the average number of detected sites was 7635 SNP sites.

Using this liquid phase chip, there were a total of 244 SNPs 75,766, with an SNP of greater than 0.05 for an MAF of 8051, with 770 sites for an MAF of greater than 0.45, and the distribution of MAFs at all sites is shown in FIG. 2, and the distribution on the chromosome is shown in FIG. 3.

The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the scope of the invention, but to limit the invention to the particular embodiments, and any modifications, equivalents, improvements, etc. that fall within the spirit and principles of the invention are intended to be included within the scope of the invention.

Claims

1. The high-oil peanut whole genome molecular marker combination is characterized by comprising 2507 high-oil gene CDS regions and/or 8003 background SNP loci; the physical locations of 2507 high oil gene CDS regions and 8003 background SNP sites were determined based on whole genome sequence alignment of the peanut reference genome; the version number of the whole genome sequence of the peanut reference genome is gca_004170445.1_asm417044v1;

2507 high oil gene CDS regions are shown as m 0001-2507;

8003 background SNP sites are shown as s 0001-8003.

2. A set of nucleotide probes for detecting the high oil peanut whole genome molecular marker combination of claim 1.

3. The nucleotide probe of claim 2, wherein the nucleotide probe comprises a combination of nucleotide probes for detecting 2507 high oil gene CDS regions; and/or nucleotide probe combinations for detecting 8003 background SNP sites.

4. A gene chip for detecting the high-oil peanut whole genome molecular marker combination according to claim 1, wherein the gene chip is a liquid phase chip and comprises a set of nucleotide probes for detecting the high-oil peanut whole genome molecular marker combination according to claim 1.

5. The gene chip of claim 4, wherein the nucleotide probes comprise a combination of nucleotide probes for detecting 2507 high oil gene CDS regions; and/or nucleotide probe combinations for detecting 8003 background SNP sites.

6. Use of the high oil peanut whole genome molecular marker combination of claim 1 or the gene chip of claim 4 or 5 in Gao Youhua genotyping.

7. Use of the high-oil peanut whole genome molecular marker combination according to claim 1 or the gene chip according to claim 4 or 5 in Gao Youhua breeding.

8. Use of the high oil peanut whole genome molecular marker combination of claim 1 or the gene chip of claim 4 or 5 in peanut oil content whole genome prediction.

9. Use of the high oil peanut whole genome molecular marker combination according to claim 1 or the gene chip according to claim 4 or 5 in Gao Youhua haplotype evaluation and identification.

10. Use of the high oil peanut whole genome molecular marker combination according to claim 1 or the gene chip according to claim 4 or 5 in high oil-based gene backcross selection.