The method is used for identifying the apos variety of the carya; the clock show is 28 apos; sNP molecular marker and application thereof

Abstract

The invention relates to the technical field of molecular markers, in particular to an SNP (Single Nucleotide Polymorphism) molecular marker and a DNA (Deoxyribose Nucleic Acid) bar code of 'Zhenxiu 28' carya illinoensis and The DNA bar code or the SNP molecular marker can be used for identifying the 'Zhenxiu 28' carya illinoensis, and is of great significance to germplasm resource identification and molecular marker breeding of the carya illinoensis.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a SNP molecular marker for identifying a thin-shell walnut variety 'Zhongxiu 28' and an application thereof. Background Art

[0002] Carya illinoinensis, also known as pecans, is known as the "longevity fruit" because its kernel is rich in unsaturated fatty acids such as oleic acid and linoleic acid, as well as proteins, polyphenols, flavonoids, trace elements and other nutrients. It has the effects of strengthening the brain, delaying aging, and preventing cardiovascular and cerebrovascular diseases, and is deeply loved by the people. With the continuous rise of the seedling consumption market, there are countless cases of "same thing with different names" and "same name with different things"; there are also cases of inferior products being sold as good ones, or even using seedlings of other species to "make up the numbers".

[0003] "Zhongxiu 28" is a new and superior variety bred by the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province. The green fruit is oblong, with a fruit shape index of 1.43. The nut is oblong, with an asymmetrical blunt tip at the top, a round base, and black stripes on the shell surface. The kernel is rich in 16.26mg / 1g of linolenic acid, significantly higher than the average level of 7.23g / 100g. The kernel is golden yellow, the shell is medium-thick, and it is easy to shell. Although "Zhongxiu 28" has more advantages and differences than other thin-shelled pecan varieties, it is still difficult for ordinary technicians to efficiently identify this variety, and the phenotypic data alone is relatively subjective, and combined with molecular biology (such as fingerprint technology) evidence will be more convincing. Molecular markers developed based on SNPs have high throughput and high sensitivity, and can be used for large-scale and accurate identification of "Zhongxiu 28". Summary of the invention

[0004] The present invention mainly aims at the above technical problems and provides a simple molecular marker method to achieve the identification of 'Zhongxiu 28' thin-shelled walnuts. Specifically, the present invention provides the following technical solutions:

[0005] In a first aspect, the present invention provides a DNA barcode for identifying or assisting in identifying the 'Zhongxiu 28' thin-shell walnut variety, characterized in that the sequence of the DNA barcode is GGGGATTT, and the positions of the bases of the DNA barcode on the chromosome are shown in the following table in order:

[0006] .

[0007] The present invention uses a high-throughput sequencing method to identify 8 SNP sites that can be used to identify or assist in identifying the 'Zhongxiu 28' thin-shell walnut variety from the whole genome of the thin-shell walnut. These SNP sites can constitute a DNA barcode unique to 'Zhongxiu 28'. It can be understood by those skilled in the art that the DNA barcode of the present invention is not composed of continuous bases, but is composed of the genotypes of SNPs dispersed at different sites on different chromosomes, so the DNA barcode of the present invention can also be called a genotype barcode.

[0008] Furthermore, the sequences of the above SNP sites are shown in SEQ ID NO.25 to SEQ ID NO.32:

[0009] .

[0010] Especially, the genotype of the locus CHR12_24676837 (chromosome 12, locus 24676837) is TT, which is different from all other varieties. Since the propagation of pecan varieties after selection is asexual propagation of single plants to ensure the stability of variety traits, the genotype of locus CHR12_24676837 TT can be used as a unique locus of 'Zhongxiu 28' and can be used for the identification or auxiliary identification of the variety.

[0011] Furthermore, the sequence of the SNP molecular marker CHR12_24676837 is shown in SEQ ID NO. 31, the SNP molecular marker CHR12_24676837 is an A / T mutation located at position 201 of SEQ ID NO.31, and the genotype of the SNP molecular marker CHR12_24676837 in the 'Zhongxiu 28' thin-shell walnut variety is TT.

[0012] On the other hand, the present invention provides KASP primers for detecting the aforementioned SNP molecular markers, wherein the primer sequences are shown in SEQ ID NOs. 1 to 24.

[0013] .

[0014] In another aspect, the present invention provides a kit for identifying or assisting in identifying the 'Zhongxiu 28' thin-shell walnut variety, wherein the kit comprises primers and / or probes for detecting the aforementioned DNA barcodes and / or SNP molecular markers.

[0015] On the other hand, another object of the present invention is to provide any of the following applications of the above-mentioned DNA barcode, molecular marker, primer, and kit:

[0016] (1) Application in identification of the thin-shelled walnut variety 'Zhongxiu 28';

[0017] (2) Application in identification, improvement or molecular marker-assisted breeding of walnut germplasm resources;

[0018] (3) Application in screening or creating different thin-shell pecan varieties;

[0019] (4) Application in constructing a DNA fingerprint database of thin-shelled pecan.

[0020] On the other hand, another object of the present invention is to provide a method for identifying or assisting in identifying the 'Zhongxiu 28' thin-shell walnut variety, characterized by comprising:

[0021] (1) extracting total DNA of the thin-shelled walnut sample to be identified;

[0022] (2) detecting the genotype of each base of the aforementioned DNA barcode;

[0023] (3) Determine the variety of the thin-shell pecan sample to be identified based on the test results.

[0024] Optionally, in step 2), the genotype of each base of the aforementioned DNA barcode can be detected by sequencing or KASP or any other available genotyping technology.

[0025] The specific judgment method of step 3) is that when the genotype of the thin-shell walnut sample to be identified is inconsistent with the aforementioned DNA barcode, it is judged to be not the 'Zhongxiu 28' thin-shell walnut variety.

[0026] Furthermore, for step 3), when the genotype of the thin-shelled walnut sample to be identified is consistent with the aforementioned DNA barcode, it is determined to be the 'Zhongxiu 28' thin-shelled walnut variety.

[0027] On the other hand, another object of the present invention is to provide another method for identifying or assisting in identifying the 'Zhongxiu 28' thin-shell walnut variety, characterized by comprising:

[0028] (1) extracting total DNA of the thin-shelled walnut sample to be identified;

[0029] (2) Detect the genotype of SNP molecular marker CHR12_24676837;

[0030] (3) Determine the variety of the thin-shell pecan sample to be identified based on the test results.

[0031] Optionally, in step 2), the genotype of the aforementioned SNP molecular marker can be detected by sequencing or KASP or any other available genotyping technology.

[0032] The specific judgment method of step 3) is that when the genotype of the thin-shell walnut sample to be identified is inconsistent with the genotype of the aforementioned SNP molecular marker CHR12_24676837, it is judged that it is not the 'Zhongxiu 28' thin-shell walnut variety.

[0033] Furthermore, in step 3), when the genotype of the thin-shelled walnut sample to be identified is consistent with the genotype of the SNP molecular marker CHR12_24676837, it is determined to be the 'Zhongxiu 28' thin-shelled walnut variety.

[0034] The aforementioned identification or auxiliary identification means directly identifying the sample to be tested as the 'Zhong Xiu 28' thin-shell pecan variety, or only excluding the sample to be identified as the 'Zhong Xiu 28' thin-shell pecan variety as an auxiliary means.

[0035] Furthermore, the aforementioned identification or auxiliary identification method can at least distinguish 'Zhongxiu 28' from any other variety described in the present invention.

[0036] In another aspect, the present invention provides any of the following applications of the aforementioned method:

[0037] (1) Application in identification of the thin-shelled walnut variety 'Zhongxiu 28';

[0038] (2) Application in identification, improvement or molecular marker-assisted breeding of walnut germplasm resources;

[0039] (3) Application in screening or creating different thin-shell pecan varieties;

[0040] (4) Application in constructing a DNA fingerprint database of thin-shelled pecan.

[0041] The present invention has the following beneficial effects: using a single DNA barcode or SNP molecular marker can effectively distinguish whether the tested variety is the 'Zhongxiu 28' thin-shelled walnut, providing a guarantee for the identification, planting, resource utilization and breeding of the 'Zhongxiu 28' thin-shelled walnut. It is also of great significance for the molecular marker breeding of thin-shelled walnuts. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The method of the present invention and its beneficial effects are described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0043] Figure 1 These are the test results of 114 samples to be tested.

[0044] Figure 2 These are the typing results of the CHR12_24676837 locus of 114 samples. The samples are arranged from left to right starting from the first row according to their numbers (there is no sample in the last black square). Figure A shows samples 1 to 94, and Figure B shows samples 95 to 114. DETAILED DESCRIPTION

[0045] 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 the embodiments. 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.

[0046] Unless otherwise defined, all technical and scientific terms used in this application have the same meanings as commonly understood by those of ordinary skill in the art to which this application belongs. The walnut plant samples used in the following examples can be obtained through commercial purchase or collected from the wild.

[0047] Example 1 Resequencing Analysis

[0048] 1. Experimental Materials

[0049] The source of materials and information are shown in Table 1:

[0050] Table 1 Sources and information

[0051]

[0052] 2. Sample DNA extraction and library construction and sequencing

[0053] First, each sample leaf was preserved in liquid nitrogen, and the genomic DNA of samples 1 to 112 was extracted using a kit. A total of 994Gb of raw data was obtained by sequencing, and the sequencing results were 150bp paired-end data.

[0054] Specific experimental steps:

[0055] The library was constructed with 1 μg DNA as the starting amount;

[0056] High-quality genomic DNA was extracted using the CTAB method;

[0057] 0.75% agarose gel electrophoresis was used to detect the size of DNA fragments and the degree of DNA degradation;

[0058] NanoDrop One spectrophotometer (Thermo Fisher Scientific) was used to detect DNA purity, with an OD260 / 280 ratio between 1.8 and 2.2, and no protein or visible impurities contamination;

[0059] The DNA concentration was detected by Qubit 3.0 fluorometer (Life Technologies, Carlsbad, CA, USA), and the detection concentration was greater than 50 ng / μl and the total amount was greater than 2 ug.

[0060] After the DNA was sheared by ultrasound using Covaris M220, magnetic beads were used for fragment selection so that the sample bands were concentrated between 200-400 bp;

[0061] The qualified libraries were arranged to be loaded onto the machine for 2*150bp sequencing.

[0062] 3. Data quality control

[0063] (1) Remove the adapter sequence from the sequence;

[0064] (2) Remove polyG and polyX at the end of the reads (minimum length is 10 bp);

[0065] (3) The average quality value of the bases in the window is calculated by counting the bases in the window in a sliding window manner, and the low-quality sliding window is trimmed. Its function is similar to Trimmomatic;

[0066] (4) Remove reads with N greater than 5;

[0067] (5) Remove reads with bases with a quality lower than 15 accounting for more than 40%;

[0068] (6) Remove reads with a length of less than 15 bp after filtering.

[0069] 5. Data comparison

[0070] In the present invention, we used the genome of Carya sylvestris as the reference genome, used BWA alignment software to align the sequenced fragments back to the reference genome, and then used Picard-tools to remove the sequenced fragments generated by PCR-duplication.

[0071] Perform bwa (version: 0.7.17; parameter: mem) alignment analysis on each sample, align the filtered clean reads to the reference genome, and calculate the alignment statistics. The specific analysis steps are as follows:

[0072] (1) Use bwa alignment software (parameter: mem -R, other parameters use software default parameters) to align the clean reads of all samples with the reference genome;

[0073] (2) Use samtools (parameter: sort) to convert the alignment results from sam (Sequence Alignment / MAP) files to sorted bam files (binary Alignment / Map);

[0074] (3) Use samtools (parameter: markdup -r) to remove duplicates from the sorted alignment results for subsequent analysis;

[0075] (4) Calculate the comparison rate and coverage.

[0076] Taking the reference genome as the standard, the average sequencing depth of the samples was 10.42× (9.29-26.08×), and the average genome coverage of all sample filtered reads after alignment to the reference genome was 85.40% (90.44 -97.44%).

[0077] A total of 31 365 448 SNP loci were developed.

[0078] Example 2 Development of core SNPs for identification of 'Jongsu 28'

[0079] 1. Identification of core SNPs

[0080] According to the grouping between 'Zhongxiu 28' and other thin-shelled walnuts, SNPs that distinguish 'Zhongxiu 28' from other thin-shelled walnuts were selected. The selection criteria were that no more than 50% of the loci of the varieties with the same genotype as 'Zhongxiu 28' could be identified after combination. At the same time, in order to reduce the false positive of SNPs, the following analysis criteria were adopted:

[0081] 1. There are no other SNPs within 30bp upstream and downstream of the SNP site;

[0082] 2. The SNP site should not have homology with other locations in the genome within 50 bp upstream and downstream;

[0083] 3. There should not be ≥8 consecutive single-base repeats within 30bp upstream and downstream of the SNP site;

[0084] 4. The GC content of 150bp before and after the selected SNP site is between 40% and 60%;

[0085] 5. Extract 250bp upstream and downstream sequences of the remaining markers to design primers, product size: 250-400; homologously align the designed primers with the genome, and filter primers that can align to multiple locations.

[0086] The remaining 8 markers were used as core SNPs, and these 8 core SNP sites were used as core SNP sites for identifying ‘Zhongxiu 28’. The genotypes of the 8 core SNP sites in different varieties were sorted from small to large according to chromosome number and position as shown in Table 2. The specific sorting order was CHR1_42893205_G_C, CHR1_57469932_G_C, CHR2_26663233_G_A, CHR3_2564907_G_A, CHR4_28328637_A_C, CHR10_6827793_T_C, CHR12_24676837_A_T, and CHR14_11071395_T_G.

[0087] The genotype combination of these 8 loci is sorted and combined into a barcode sequence of GGGGATTT in 'Zhongxiu 28'. The SNP genotype combination of these 8 loci is completely different from that of other varieties (Table 2), and can be used as a DNA barcode for identifying 'Zhongxiu 28'. In particular, the genotype of the locus CHR12_24676837 is TT, which is different from all other varieties. Considering that the propagation of pecan varieties after selection is a single plant asexual reproduction to ensure the stability of variety traits. Therefore, the genotype of the locus CHR12_24676837 is TT, which can be considered as a unique locus of 'Zhongxiu 28' and can be used for the identification of this variety.

[0088]

[0089] Example 3 Molecular marker verification

[0090] 1) Extracting total DNA from samples to be tested (independently verifying that the sample variety number is consistent with Example 1, and adding non-thin-shell pecan samples No. 115-114);

[0091] 2) Primer and probe design;

[0092] According to the SNP sites determined in Example 1, KASP primers and probes were designed based on the chromosome sequence. The primer and probe sequences are shown in Table 3.

[0093]

[0095] 3) Using the primers and probes designed in step 2) to perform KASP detection and genotyping on the qualified DNA extracted in step 1). The primers and probes are combined to prepare a detection kit.

[0096] 4) Determine the type of sample based on the mass spectrometry results.

[0097] Results Figure 1~2 , Figure 1It shows that sample No. 80 is the variety 'Zhongxiu 28', and the genotype sequence of its 8 loci is GGGGATTT, which is completely different from the genotypes of other samples. Figure 2 The results showed that the CHR12_24676837 locus of 'Zhongxiu 28' variety was TT, which was completely different from other samples.

[0098] In summary, the DNA barcode prepared by the present invention can be used for accurate identification of the 'Zhong Xiu 28' variety, and the single SNP site CHR12_24676837 can directly identify the 'Zhong Xiu 28' variety.

[0099] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to the above embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A SNP molecular marker for identifying the 'Zhongxiu 28' thin-shelled walnut variety, characterized in that: The SNP molecular marker includes SNP molecular marker CHR12_24676837, the sequence of the SNP molecular marker CHR12_24676837 is shown in SEQ ID NO.31, the mutation type of the SNP molecular marker CHR12_24676837 is an A / T mutation located at position 201 on SEQ ID NO.31, and the genotype of the SNP molecular marker CHR12_24676837 in the 'Zhongxiu 28' thin-shell walnut variety is TT.

2. A SNP molecular marker for identifying the 'Zhongxiu 28' thin-shelled walnut variety, The sequences of the SNP molecular markers are shown in SEQ ID NO.25 to SEQ ID NO.32, and the SNP molecular markers are shown in the following table in order: 。 3. KASP primers for detecting the SNP molecular markers according to claim 1 or claim 2.

4. A kit for identifying the 'Zhongxiu 28' thin-shelled walnut variety, the kit comprising primers and / or probes for detecting the SNP molecular markers described in claim 1 or claim 2.

5. Any of the following uses of the molecular marker according to claim 1, the molecular marker according to claim 2, the primer according to claim 3 or the kit according to claim 4: (1) Application in identification of the thin-shelled walnut variety 'Zhongxiu 28'; (2) Application in identification, improvement or molecular marker-assisted breeding of walnut germplasm resources; (3) Application in screening or creating different thin-shell pecan varieties; (4) Application in constructing a DNA fingerprint database of thin-shelled pecan.

6. A method for identifying the 'Zhongxiu 28' thin-shell walnut variety, characterized in that: include: (1) extracting total DNA of the thin-shelled walnut sample to be identified; (2) detecting the genotype of the SNP molecular marker described in claim 1; (3) Determine the variety of the thin-shell pecan sample to be identified based on the test results.

7. The method according to claim 6, characterized in that Step 2) The genotype of the SNP molecular marker of claim 1 can be detected by sequencing or KASP or any other available genotyping technology.

8. A method for identifying the 'Zhongxiu 28' thin-shell walnut variety, characterized in that: include: (1) extracting total DNA of the thin-shelled walnut sample to be identified; (2) detecting the genotype of the SNP molecular marker described in claim 2; (3) Determine the variety of the thin-shell pecan sample to be identified based on the test results.

9. The method according to claim 8, characterized in that Step 2) The genotype of the SNP molecular marker of claim 2 can be detected by sequencing or KASP or any other available genotyping technology.

10. Any of the following applications of the method according to any one of claims 6 to 9: (1) Application in identification of the thin-shelled walnut variety 'Zhongxiu 28'; (2) Application in identification, improvement or molecular marker-assisted breeding of walnut germplasm resources; (3) Application in screening or creating different thin-shell pecan varieties; (4) Application in constructing a DNA fingerprint database of thin-shelled pecan.

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