Alfalfa liquid phase breeding chip

Abstract

The present invention discloses an alfalfa liquid-phase breeding chip. Specifically, the present invention discloses the use of 5037 SNP sites of alfalfa or a substance for detecting the 5037 SNP sites in any of the following: (1) preparing a kit for detecting the genotype of alfalfa SNP sites; (2) alfalfa molecular breeding; (3) constructing a DNA fingerprint database of alfalfa germplasm resources or varieties; and (4) detecting the similarity between any two alfalfa varieties. By comparing the 5037 SNP sites of alfalfa of the present invention, it is possible to realize molecular breeding of alfalfa, construct a DNA fingerprint database of alfalfa germplasm resources or varieties, and detect the similarity between any two alfalfa varieties, thereby realizing cluster analysis of alfalfa.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology and relates to an application of 5037 SNP sites in alfalfa and an alfalfa liquid-phase breeding chip based on the 5037 SNP sites. Background Art

[0002] Alfalfa, a perennial herbaceous plant of the genus Medicago in the Leguminosae family, has erect, tufted, or prostrate stems that are quadrangular and heavily branched. Its stipules are large, ovate-lanceolate, and its leaflets are obovate-oblong. Its flowers form family-like racemes, with bell-shaped pedicels and purple corollas. Its fruit is spiral-shaped and brown when ripe. Its seeds are small, smooth, and yellow or brown. It blooms from May to July, and fruiting occurs from June to August. The name "alfalfa" comes from its purple flowers.

[0003] Prior art research on alfalfa germplasm resources typically involves systematically documenting the resource material's growth period, morphological characteristics, pest and disease resistance, and adaptability to the climatic environment. Field observations then identify desirable and unique traits, leading to genetic studies. This research process is lengthy, subject to significant human influence, and results in limited accuracy. Summary of the Invention

[0004] The object of the present invention is to provide an application of 5037 SNP sites in alfalfa and an alfalfa liquid-phase breeding chip based on the 5037 SNP sites.

[0005] The present invention claims the use of 5037 SNP sites or substances for detecting the 5037 SNP sites in any of the following applications:

[0006] (1) Preparing a kit for detecting the genotype of alfalfa SNP sites;

[0007] (2) Molecular breeding of alfalfa;

[0008] (3) Construct a DNA fingerprint database of alfalfa germplasm resources or varieties;

[0009] (4) Detect the similarity between any two alfalfa varieties;

[0010] The physical locations of the 5037 SNP sites are determined based on the whole genome sequence alignment of the alfalfa variety Zhongmu 4. The version number of the whole genome sequence of the alfalfa variety Zhongmu 4 is GWH:GWHBECI00000000, and the upload date is the Zhongmu-4 whole gene sequence on August 21, 2021; the 5037 SNP sites are 584 sites located on chromosome 1; 558 sites located on chromosome 2; 666 sites located on chromosome 3; 637 sites located on chromosome 4; 546 sites located on chromosome 5; 737 sites located on chromosome 6; 607 sites located on chromosome 7; and 702 sites located on chromosome 8.

[0011] Among them, the 5037 SNP sites are specifically shown in Table 1 in the embodiment, where SNP-id represents the number of the SNP site, chrom represents the chromosome number where the site is located, position represents the position of the site on the chromosome, and ref and alt represent the two types of each SNP site.

[0012] The present invention provides a kit for detecting SNP genotypes in alfalfa, comprising a substance for detecting the 5037 sites. The genotype detection kit can be a substance for sequencing the genome of the plant to be tested and then performing data comparison based on the sequencing results; or it can be a liquid-phase gene chip designed for the 5037 sites.

[0013] The invention provides a method for detecting alfalfa germplasm, comprising the steps of extracting whole genome DNA of the alfalfa to be detected and detecting the genotype of the 5037 sites.

[0014] The present invention provides a method for cluster analysis of alfalfa, comprising:

[0015] 1) extracting whole genome DNA of alfalfa to be tested and detecting the genotypes of 5037 loci therein;

[0016] 2) Building a cluster dendrogram based on the obtained genotype data, thereby dividing the alfalfa into different groups.

[0017] The present invention provides a method for constructing an alfalfa DNA fingerprint database, comprising the following steps:

[0018] (a1) extracting genomic DNA of all alfalfa varieties used to construct an alfalfa DNA fingerprint database;

[0019] (a2) detecting the genotypes of the 5037 SNP sites in the genomic DNA of all alfalfa varieties, and the obtained genotype data constitutes an alfalfa DNA fingerprint database.

[0020] The present invention provides an alfalfa molecular breeding method, comprising the following steps: hybridizing an alfalfa donor parent carrying a target trait with an alfalfa recipient parent, then backcrossing the obtained hybrid offspring with the alfalfa donor parent several times, respectively detecting the genotypes of 5037 SNP sites in the genomic DNA of the alfalfa recipient parent and the obtained backcross offspring carrying the target trait, and selecting, from the backcross offspring carrying the target trait, individuals whose genotypes at the 5037 SNP sites are most consistent with those of the alfalfa recipient parent based on the detection results.

[0021] The beneficial effects of the present invention are that, by comparing the 5037 SNP sites of alfalfa of the present invention, it is possible to realize molecular breeding of alfalfa, construct a DNA fingerprint database of alfalfa germplasm resources or varieties, and detect the similarity between any two alfalfa varieties, thereby realizing cluster analysis of alfalfa. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The distribution of 5037 SNP sites on 8 chromosomes.

[0023] Figure 2 Schematic diagram of the results of dividing 334 alfalfa species into 4 subgroups.

[0024] Figure 3 This is a screenshot of the webpage of the full genome sequence version of the alfalfa variety Zhongmu 4 of the present invention. DETAILED DESCRIPTION

[0025] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0026] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0027] The following examples of Zhongmu No. 1, Zhongmu No. 3, Zhongmu No. 4, and Zhongmu No. 5 are disclosed in the comparative study of the production performance of 22 alfalfa varieties in Hebei Province, Lv Huigang, Acta Grasslanda Sinica, July 2018; Genome Assembly of Alfalfa Cultivar Zhongmu-4 and Identification of SNPs Associated with Agronomic Traits; Effect of Seeding Rate and Row Spacing Configuration on Alfalfa Yield and Quality in Saline-alkali Land; etc. The public can obtain this material from the Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences for repeating this experiment.

[0028] Example 1

[0029] The VCF file was obtained using the resequencing data of 220 alfalfa materials, with the alfalfa reference genome zhongmu-4 as the reference (the version number of the full genome sequence of the alfalfa variety Zhongmu-4 is GWH:GWHBECI00000000, and the upload date is the full genome sequence of Zhongmu-4 on August 21, 2021. The webpage screenshot is as follows Figure 3 As shown), the target segments are preliminarily screened for the site information in the data, and then the probes corresponding to the target segments are synthesized. By selecting 220 alfalfa varieties for chip data testing, the target areas with good uniformity are preferentially selected, and the overall uniform distribution is guaranteed. Finally, 5037 SNP sites were screened, including 94 sites related to basic agronomic traits, salt tolerance and quality traits. These sites are distributed as follows: 584 sites on chromosome 1; 558 sites on chromosome 2; 666 sites on chromosome 3; 637 sites on chromosome 4; 546 sites on chromosome 5; 737 sites on chromosome 6; 607 sites on chromosome 7; 702 sites on chromosome 8, as shown Figure 1 shown.

[0030] The positions of the 5037 SNP sites in each chromosome and their genotypes in zhongmu4 are shown in Table 1, where SNP-id represents the number of the SNP site, chrom represents the chromosome number where the site is located, position represents the position of the site on the chromosome, ref and alt represent the two types of each SNP site, and del represents deletion.

[0031] Table 1

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[0036]

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[0065]

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[0070]

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[0090]

[0091]

[0092] Example 2

[0093] Based on the aforementioned 5037 SNPs, this embodiment provides a kit for detecting SNP genotypes in alfalfa, including materials for detecting the 5037 SNPs. The genotype detection kit can be a kit for sequencing the genome of the plant to be tested, followed by data comparison based on the sequencing results, or a liquid-phase gene chip designed for the 5037 SNPs.

[0094] This embodiment also provides a method for detecting alfalfa germplasm, comprising the steps of extracting whole genome DNA of the alfalfa to be tested and detecting the genotype of the 5037 loci.

[0095] This embodiment also provides a method for cluster analysis of alfalfa, comprising:

[0096] 1) extracting whole genome DNA of alfalfa to be tested and detecting the genotypes of 5037 loci therein;

[0097] 2) Building a cluster dendrogram based on the obtained genotype data, thereby dividing the alfalfa into different groups.

[0098] This embodiment also provides a method for constructing an alfalfa DNA fingerprint database, comprising the following steps:

[0099] (a1) extracting genomic DNA of all alfalfa varieties used to construct an alfalfa DNA fingerprint database;

[0100] (a2) detecting the genotypes of the 5037 SNP sites in the genomic DNA of all alfalfa varieties, and the obtained genotype data constitutes an alfalfa DNA fingerprint database.

[0101] This embodiment can also provide an alfalfa molecular breeding method, comprising the following steps: hybridizing an alfalfa donor parent carrying a target trait with an alfalfa recipient parent, then backcrossing the resulting hybrid offspring with the alfalfa donor parent several times, detecting the genotypes of the 5037 SNP sites in the genomic DNA of the alfalfa recipient parent and the resulting backcross offspring carrying the target trait, and selecting, based on the test results, individuals from the backcross offspring carrying the target trait whose genotypes at the 5037 SNP sites are most consistent with those of the alfalfa recipient parent.

[0102] Example 3334 Detection of Alfalfa Varieties

[0103] 1. 334 alfalfa samples belonging to Zhongmu No. 1, Zhongmu No. 3, Zhongmu No. 4 and Zhongmu No. 5 were tested for varieties according to the following method.

[0104] 1. Extract genomic DNA from samples and construct sample libraries;

[0105] 1) Sample DNA extraction

[0106] The sample DNA was extracted using the CTAB method.

[0107] 2) Sample DNA quality inspection

[0108] The DNA concentration of the test sample was determined using Qubit Fluorometric Quantitation (Thermo Fisher), and the integrity of the DNA was checked using 1% agarose gel electrophoresis. The qualified samples were placed in a 4°C refrigerator for storage and future use.

[0109] 3) Sample DNA fragmentation

[0110] Take 12 μL of DNA that has passed the quality inspection and place it in a 0.2 μL PCR tube. Place the tube in an ultrasonic crusher to randomly physically break the DNA into fragments of 200 to 400 bp.

[0111] 4) Sample end repair

[0112] Add 4 μL of GenoBaits End Repair Buffer (GenoBaits, Shijiazhuang Boruidi Biotechnology Co., Ltd.) and 2.7 μL of GenoBaits End Repair Enzyme to the tube, add water to 20 μL, and place it in an ABI 9700 PCR instrument and incubate at 37°C for 20 minutes to complete the end repair and A addition process of the broken fragments.

[0113] 5) Sample sequencing adapter connection

[0114] Remove the tube from the PCR instrument and add 2μL GenoBaits Ultra DNA ligase, 8μL GenoBaits Ultra DNA Ligase Buffer and 2μL GenoBaits Adapter, add water to 40μL, and then place it on the ABI9700 PCR instrument at 22℃ for 30 minutes to complete the connection of the sequencing adapter.

[0115] 6) Sample DNA purification

[0116] 48 μL of Beackman AMPure XP Beads (Beackman) were added to the ligation product to purify the ligation product. After purification, magnetic beads were used for fragment screening, and ligation products with insert fragments between 200 and 300 bp were retained.

[0117] 7) Sample library amplification

[0118] To the PCR tube from the previous step, add 5 μL of sequencing adapters with barcode sequences, 1 μL of P5 adapters, and 10 μL of GenoBaits PCR Master Mix. Make up to 20 μL with purified water. Amplify using an ABI 9700 PCR instrument using the following program: pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds. Repeat steps 2-4 for a total of 8 cycles. Finally, extend at 72°C for 5 minutes. Different barcodes are used to distinguish different samples.

[0119] 8) Sample library purification

[0120] Add 24 μL of Beckmen AMPure XP Beads (Beackman) to the second-round PCR product, pipette up and down to mix evenly, place a 0.2 μL PCR tube on a magnetic stand until the solution is clear, discard the supernatant, wash the magnetic beads once with 75% ethanol, and elute the library DNA with Tris-HCl (pH 8.0).

[0121] 2. Determine the genotype of the target plant at 5037 SNP sites using a liquid-phase gene chip manufactured by Boridi or determine the genotype of the 5037 sites described in Example 1 by gene sequencing;

[0122] 1) DNA hybridization

[0123] Take 500 ng of the constructed genomic DNA sequencing library, add 5 μL of GenoBaits Block I and 2 μL of GenoBaits Block II, place it on an Eppendorf Concentrator plus (Eppendorf), and evaporate it to dry powder at ≤70°C. Add 8.5 μL of GenoBaits 2x Hyb Buffer, 2.7 μL of GenoBaits Hyb Buffer Enhancer, and 2.8 μL of Nuclease-Free Water to the dry powder tube, mix thoroughly with a pipette, and incubate it on an ABI 9700 PCR instrument at 95°C for 10 minutes. Then, remove the PCR tube and add 3 μL of the synthesized probe (probe concentration is 60 ng / μL). Vortex to mix thoroughly, and incubate it on an ABI 9700 PCR instrument at 65°C for 2 hours to complete the probe hybridization reaction.

[0124] 2) DNA capture

[0125] Add 100 μL of GenoBaits DNA Probe Beads to the reaction system after hybridization in the previous step, pipette up and down 10 times, and incubate at 65°C on an ABI 9700 PCR instrument for 45 minutes to allow the magnetic beads to bind to the probe. Wash the probe-bound magnetic beads at 65°C with 100 μL of GenoBaits Wash Buffer I and 150 μL of GenoBaits Wash Buffer II, respectively. Then, wash the magnetic beads at room temperature with 100 μL of GenoBaits Wash Buffer I, 150 μL of GenoBaits Wash Buffer II, and 150 μL of GenoBaits Wash Buffer III, respectively. Resuspend the washed magnetic beads in 20 μL of Nuclease-Free Water.

[0126] Take 13 μL of resuspended DNA (with magnetic beads) and add it to a new 0.2 mL PCR tube. Then add 15 μL GenoBaits PCR Master Mix and 2 μL GenoBaits Primer Mix to prepare the post-PCR system. Use ABI9700 PCR instrument to amplify the library. The amplification program is as follows: pre-denaturation at 95 ° C for 5 min, denaturation at 95 ° C for 30 s, annealing at 60 ° C for 30 s, and extension at 72 ° C for 30 s; repeat steps 2-4 for a total of 15 cycles; extension at 72 ° C for 5 min.

[0127] Add 45 μL of Beckman AMPure XP Beads (Beackman) to the post-PCR product and pipette up and down to evenly distribute the solution. Place the 0.2 mL PCR tube on a magnetic stand until the solution is clear. Discard the supernatant, wash the beads twice with 75% ethanol, and elute the library DNA with Tris-HCl (pH 8.0). This completes the probe hybridization capture process.

[0128] 3) DNA hybridization capture library quality inspection

[0129] The DNA concentration of the library was determined using Qubit Fluorometric Quantitation (Thermo Fisher), and then agarose gel electrophoresis was used to detect whether the fragment size of the library DNA was between 300 and 400 bp.

[0130] 4) DNA hybridization capture library sequencing

[0131] The constructed DNA library was sequenced using the BGI MGISEQ2000 sequencer.

[0132] 5) Genotype data analysis

[0133] After quality control by FastQC (www.bioinformatics.babraham.ac.uk / project), the sequencing data were aligned to the reference genome using the default parameters of BWA (bio-bwa.sourceforge.net). SNPs were identified in the sequencing data using GATK (software.broadinstitute.org / gatk) software, and genotyping information from probe capture sequencing was extracted using a self-written Perl script to generate the final genotyping file.

[0134] 2. Cluster analysis of 334 alfalfa samples

[0135] The genotypes of 334 alfalfa varieties at 5037 SNP sites and their detection rates were compared, and the results are shown in Tables 2 and Figure 2 As shown in Table 2, the genotyping results showed that the detection rate of 5037 SNP sites in 334 alfalfa varieties ranged from 97.20% to 99.88%.

[0136] Table 2

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[0146] 3. Group structure of 334 materials

[0147] According to the test results in step 1, the 334 materials were divided into groups and found that these materials can be divided into 4 subgroups. The results are as follows Figure 2 As shown. Figure 2 It can be seen that the 334 alfalfa samples are divided into four categories, which correspond one-to-one to their sources, Zhongmu No. 1, Zhongmu No. 3, Zhongmu No. 4 and Zhongmu No. 5.

[0148] The above examples show that the comparison of 5037 SNP sites of alfalfa of the present invention can realize molecular breeding of alfalfa, construction of DNA fingerprint database of alfalfa germplasm resources or varieties, and detection of similarity between any two alfalfa varieties, thereby realizing cluster analysis of alfalfa.

[0149] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. Use of a substance for detecting genotypes at 5037 loci in any of the following: (1) Construct a DNA fingerprint database of alfalfa germplasm resources; (2) testing the similarity of any two alfalfa varieties; The physical locations of the 5037 sites are determined based on the whole genome sequence alignment of the alfalfa variety Zhongmu-4, whose version number is GWH:GWHBECI00000000 and whose upload date is August 21, 2021. The specific locations of the 5037 sites are shown in the following table: Wherein SNP-id represents the number of the site, chrom represents the chromosome number where the site is located, position represents the position of the site on the chromosome, ref and alt represent two types of each site, and the alfalfa is Zhongmu No. 1, Zhongmu No. 3, Zhongmu No. 4 or Zhongmu No.

5.

2. A method for detecting alfalfa germplasm, characterized in that: The method comprises the steps of extracting the whole genome DNA of the alfalfa to be tested and detecting the genotype of the 5037 sites described in claim 1, wherein the alfalfa is Zhongmu No. 1, Zhongmu No. 3, Zhongmu No. 4 or Zhongmu No.

5.

3. A method for cluster analysis of alfalfa, characterized in that: include: 1) extracting whole-genome DNA of alfalfa to be tested and detecting the genotype of the 5037 loci described in claim 1; 2) A cluster dendrogram is established based on the obtained genotype data, thereby dividing the alfalfa into different groups, and the alfalfa is Zhongmu No. 1, Zhongmu No. 3, Zhongmu No. 4 or Zhongmu No.

5.

4. A method for constructing an alfalfa DNA fingerprint database, comprising the following steps: (a1) extracting genomic DNA of all alfalfa varieties used to construct an alfalfa DNA fingerprint database; (a2) detecting the genotypes of the 5037 sites described in claim 1 in the genomic DNA of all the alfalfa varieties, and the obtained genotype data constitute an alfalfa DNA fingerprint database, wherein the alfalfa is Zhongmu No. 1, Zhongmu No. 3, Zhongmu No. 4 or Zhongmu No. 5.

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