The method is used for identifying the apos variety of the carya; the Yao gui No. 3 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 of Yaolu No.3 carya illinoensis and application of the SNP molecular marker. The SNP molecular marker disclosed by the invention can be used for identifying the 'Yaolu No.3' carya illinoensis, and has important significance on 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 'Yaogou No. 3' and an application thereof. Background Art

[0002] Carya illinoinensis, a large deciduous tree of the genus Carya in the family Juglandaceae, is also known as the American pecan, and is native to the Americas. Carya illinoinensis is a world-renowned ecological and economic tree species with delicious and non-astringent fruits, high yield, straight trunk, and excellent wood. The kernel is rich in unsaturated fatty acids such as oleic acid and linoleic acid, as well as various nutrients such as protein, polyphenols, flavonoids, and trace elements. It has the effects of strengthening the brain, delaying aging, and preventing cardiovascular and cerebrovascular diseases. It is also known as the longevity fruit and is deeply loved by the people. In recent years, with the breakthrough of breeding technology, the planting area has been continuously expanded; however, there are also numerous 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". How to identify and identify seedling varieties is a problem that needs to be solved in the current promotion and application of carya illinoinensis seedlings.

[0003] "Yaogou No. 3" is a new superior variety selected by the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province. The fruit matures in mid-October. The nut is oblong, with an asymmetrical sharp angle on the top and a round base. The surface of the shell has brown-black to black stripes and many spots. The average weight of the nut is 8.41 g, the longitudinal diameter is 39.98 mm, the transverse diameter is 23.00 mm, and the fruit shape index is 1.842. The average kernel content is 55.67%, and the oil content of the seed kernel is 73.82% (the highest oil content under the same measurement conditions). The unsaturated fatty acid content in the seed kernel is 376.47 mg / g, accounting for 93.16% of the total fatty acid content, of which monounsaturated fatty acids account for 72.60% (the content of monounsaturated fatty acids in the seed kernel is high), and polyunsaturated fatty acids account for 27.40%. The seed kernel is golden yellow and very full; the shell is medium thick, about 0.748 mm thick, and easy to shell. Although "Yaogou No. 3" has many advantages and differences compared with other thin-shelled pecan varieties, it is still difficult for ordinary technicians to efficiently identify this variety. Moreover, phenotypic data alone is relatively subjective, and combined with molecular biology (such as fingerprint technology) evidence will be more convincing. Although there are some molecular markers for pecans in the existing technology, most of them are limited to AFLPs and SSR. And there are no reports on molecular markers for the identification of "Yaogou No. 3". The DNA barcode developed based on molecular marker technology has a large throughput and high sensitivity, and can be used for large-scale and accurate identification of "Yaogou No. 3". 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 'Yaogou No. 3' thin-shelled walnuts. Specifically, the present invention provides the following technical solutions:

[0005] In a first aspect, the present invention provides a SNP molecular marker for identifying or assisting in identifying the 'Yaogou No. 3' thin-shelled walnut variety, characterized in that the positions of the bases of the SNP molecular marker on the chromosome are shown in the following table in order:

[0006] .

[0007] The present invention uses a high-throughput sequencing method to identify 7 SNP sites that can be used to identify or assist in identifying the 'Yaogou No. 3' thin-shell walnut variety from the whole genome of the thin-shell walnut, and these SNP sites can constitute a DNA barcode unique to 'Yaogou No. 3'. 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 of genotypes 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 SNP sites in the above DNA barcodes are shown in SEQ ID NO.22 to SEQ ID NO.28 (in the order of the positions of the SNPs in the DNA barcodes):

[0009]

[0010] In particular, the genotype of the locus CHR5_2793044 (chromosome 5, locus 2793044) is GG, which is different from all other varieties. Since the propagation of pecan varieties after selection is asexual reproduction of a single plant to ensure the stability of the variety's traits, the genotype of the locus CHR5_2793044 is GG, which can be used as a unique locus of 'Yaogou No. 3' and can be used for the identification or auxiliary identification of the variety.

[0011] Furthermore, the sequence of the SNP molecular marker CHR5_2793044 is shown in SEQ ID NO.25, the SNP molecular marker CHR5_2793044 is an A / G mutation located at position 201 of SEQ ID NO.25, and the genotype of the SNP molecular marker CHR5_2793044 in the 'Yaogou No. 3' thin-shell walnut variety is GG.

[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. 1-21.

[0013] .

[0014] On the other hand, the present invention provides a kit for identifying or assisting in identifying the 'Yaogou No. 3' thin-shelled walnut variety, the kit comprising 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 'Yaogou No. 3';

[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 'Yaogou No. 3' 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 'Yaogou No. 3' thin-shell walnut variety.

[0026] Further, 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 'Yaogou No. 3' 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 'Yaogou No. 3' 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 CHR5_2793044;

[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 CHR5_2793044, it is judged that it is not the 'Yaogou No. 3' thin-shell walnut variety.

[0033] Furthermore, in step 3), when the genotype of the thin-shell walnut sample to be identified is consistent with the genotype of the SNP molecular marker CHR5_2793044, it is determined to be the 'Yaogou No. 3' thin-shell walnut variety.

[0034] The aforementioned identification or auxiliary identification means directly identifying the sample to be tested as the 'Yaogou No. 3' thin-shelled walnut variety, or only excluding the sample to be identified as the 'Yaogou No. 3' thin-shelled walnut variety as an auxiliary means.

[0035] Furthermore, the aforementioned identification or auxiliary identification method can at least distinguish 'Yaogou No. 3' 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 'Yaogou No. 3';

[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: a single DNA barcode or SNP molecular marker can be used to effectively distinguish whether the tested variety is the 'Yaogou No. 3' thin-shelled walnut, providing a guarantee for the identification, planting, resource utilization and breeding of the 'Yaogou No. 3' 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 CHR5_2793044 locus of 114 samples. The samples are arranged from left to right starting from the first row according to the order of 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 Material sources and information (Note: the same sample has different names)

[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] 4. 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 'Yaogou No. 3'

[0079] 1. Identification of core SNPs

[0080] According to the grouping between 'Yaogou No. 3' and other thin-shelled walnuts, SNPs were selected to distinguish 'Yaogou No. 3' from other thin-shelled walnuts. The selection criteria were that no more than 50% of the loci had the same genotype as 'Yaogou No. 3', and that 'Yaogou No. 3' 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] Finally, the 7 markers selected were used as core SNPs, and these 7 core SNP loci were used as core SNP loci for identifying 'Yaogou No. 3'. The genotypes of the 7 core SNP loci 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 CHR2_26663233, CHR3_2564907, CHR4_5937447, CHR5_2793044, CHR5_10451065, CHR11_4001936, and CHR12_24676837.

[0087] The genotype combination of these 7 loci is sorted and combined into a barcode sequence of RAMGCCW in 'Yaogou No. 3'. The genotype of these 7 loci is completely different from the SNP genotype combination of other varieties (Table 2), and can be used as a DNA barcode for identifying 'Yaogou No. 3'. In particular, the genotype of the locus CHR5_2793044 is GG, which is different from all other varieties. Considering that the expansion of pecan varieties after selection is asexual reproduction of a single plant to ensure the stability of variety traits. Therefore, the GG genotype of the locus CHR5_2793044 can be considered as a unique locus of 'Yaogou No. 3' and can be used for the identification of this variety.

[0088] Table 2 Multiple sequence alignment results of the 114 sample SNP sites arranged in ascending order of SNP numbers

[0089]

[0090] Example 3 Molecular marker verification

[0091] 1) Extract the total DNA of the sample to be tested (independently verify that the sample variety number is consistent with Example 1, and add non-thin-shell pecan species samples No. 113-114);

[0092] 2) Primer and probe design;

[0093] 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.

[0094] Table 3 Kasp primers

[0095]

[0096] 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.

[0097] 4) Determine the type of sample based on the mass spectrometry results.

[0098] Results Figure 1~2 , Figure 1 The results showed that the sample No. 47 was of the variety 'Yaogou No. 3', and the genotype sequence of its 7 loci was RAMGCCW, which was completely different from the genotypes of other samples. Figure 2 The results showed that the CHR5_2793044 locus of the 'Yaogou No. 3' variety was GG, which was completely different from the other samples.

[0099] In summary, the DNA barcode prepared by the present invention can be used for accurate identification of the 'Yaogou No. 3' variety, and the single SNP site CHR5_2793044 can directly identify the 'Yaogou No. 3' variety.

[0100] 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 'Yaogou No. 3' thin-shelled walnut variety, characterized in that: The SNP molecular marker includes molecular marker CHR5_2793044, the sequence of the SNP molecular marker CHR5_2793044 is shown in SEQ ID NO.25, the variation type of the SNP molecular marker CHR5_2793044 is an A / G mutation located at position 201 on SEQ ID NO.25, and the genotype of the molecular marker CHR5_2793044 in the 'Yaogou No. 3' thin-shell walnut variety is GG.

2. A SNP molecular marker for identifying the 'Yaogou No. 3' thin-shelled walnut variety, characterized in that , the sequences of the SNP molecular markers are shown in SEQ ID NO.22 to SEQ ID NO.28, and the SNP molecular markers are shown in the following table in order and mutation type: 。 3. A KASP primer for detecting the molecular marker according to claim 1 or claim 2, characterized in that: The primers can amplify and detect the SNP molecular markers described in claim 1 or claim 2.

4. A kit for identifying the 'Yaogou No. 3' thin-shelled walnut variety, the kit comprising primers and / or probes for detecting the SNP molecular markers of claim 1 and / 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 'Yaogou No. 3'; (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 'Yaogou No. 3' thin-shelled 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) detecting the genotype of the SNP molecular marker of claim 1 by sequencing or KASP or any other available genotyping technology.

8. A method for identifying the 'Yaogou No. 3' thin-shelled 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) detecting the genotype of the SNP molecular marker of claim 2 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 'Yaogou No. 3'; (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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