KASP primer for identifying different carya illinoensis varieties and application

Abstract

The invention relates to the technical field of molecular markers, in particular to KASP primers of 112 carya illinoensis varieties, an SNP molecular marker, a fingerprint spectrum and application of the KASP primers and the SNP molecular marker. By adopting the technical scheme provided by the invention, the carya illinoensis fingerprint spectrum can be drawn, is used for accurate identification of carya illinoensis varieties, and has important significance on germplasm resource identification and molecular marker breeding of carya illinoensis.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to KASP primers, SNP molecular markers, fingerprints and applications thereof for identifying different walnut varieties. Background Art

[0002] Thin-shell pecan (Carya illinoinensis (Wangenh.) K.Koch), a deciduous tree of the genus Carya in the Juglandaceae family, has kernels 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.

[0003] In recent years, with the breakthrough of breeding technology, the seedling sales market has been heating up. However, there are countless cases of "same thing with different names" and "same name with different things" in the market; inferior products are often sold as good ones, or even seedlings of other species are used as "fake products".

[0004] Competitive allele specific PCR (KASP) is a new genotyping technology based on single nucleotide polymorphisms (SNPs), which can accurately type SNPs and insertion-deletion polymorphisms (Indels) at the genomic level. KASP genotyping technology can be used for high-throughput, low-cost, rapid and simple verification, and the selected markers can be used in variety identification and differentiation as well as molecular marker-assisted breeding. KASP technology has been widely used in molecular marker-assisted selection breeding of various crops, but it has not been reported in thin-shelled pecans. Although there are some molecular markers about pecans in the existing technology, most of them are limited to AFLPs and SSRs. Collecting and organizing the core germplasm resources of thin-shelled pecans, developing new core SNP molecular markers that can be used for thin-shelled pecan germplasm identification, and constructing fingerprint maps can establish variety labels for large populations of thin-shelled pecans. Summary of the invention

[0005] The present invention mainly aims at the above technical problems, provides a simple molecular marker method, constructs a fingerprint of thin-shelled walnuts, and realizes the identification of 112 different varieties of thin-shelled walnuts. Specifically, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides a SNP molecular marker combination for identifying or assisting in identifying a thin-shell pecan variety, characterized in that the position of the SNP molecular marker on the chromosome is as shown in Table 1:

[0007] Table 1 Location of SNP markers on chromosomes .

[0008] The present invention utilizes a high-throughput sequencing method to identify 33 SNP sites in the whole genome of thin-shell walnuts that can be used to identify or assist in identifying thin-shell walnut varieties. These SNP sites can form a fingerprint map for the identification of 112 types of thin-shell walnuts.

[0009] Furthermore, the sequence of the SNP site in the above fingerprint map is shown as SEQ ID NO.100~SEQ ID NO.132, and the SNP is located at the 201st position of any sequence of SEQ ID NO.100~SEQ ID NO.132.

[0010] On the other hand, a fingerprint map for identifying thin-shelled pecan varieties is provided, characterized in that the fingerprint map is composed of the aforementioned SNP molecular markers, the sequence of the SNP molecular markers is shown in SEQ ID NO.100~SEQ ID NO.132, and the SNP is located at the 201st position of SEQ ID NO.100~SEQ ID NO.132.

[0011] Those skilled in the art can understand that the fingerprint map described in the present invention is not composed of continuous bases, but is composed of the genotypes of SNPs scattered at different sites on different chromosomes. Therefore, the genotypes of the 33 SNP sites of different varieties of the present invention can constitute different genotype barcodes, and the fingerprint map can also be considered to be composed of the genotype barcodes of different varieties of thin-shell pecans.

[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 NO.1 to SEQ ID NO.99.

[0013] On the other hand, the present invention provides a kit for identifying or assisting in identifying a thin-shell pecan variety, the kit comprising primers for detecting the aforementioned SNP molecular markers and / or fingerprints.

[0014] On the other hand, another object of the present invention is to provide any of the following applications of the above-mentioned SNP molecular markers, fingerprints, primers, and kits:

[0015] (1) Application in identification of thin-shell pecan varieties;

[0016] (2) Application in identification, improvement or molecular marker-assisted breeding of walnut germplasm resources;

[0017] (3) Application in screening or creating different thin-shell pecan varieties;

[0018] (4) Application in constructing a DNA fingerprint database of thin-shelled pecan.

[0019] On the other hand, another object of the present invention is to provide a method for constructing a fingerprint of thin-shelled walnuts, characterized in that the method comprises the following steps:

[0020] (1) Extracting total DNA from samples of different varieties of thin-shelled pecans;

[0021] (2) detecting the genotype of the SNP molecular marker in the sample, wherein the sequence of the SNP molecular marker is shown in SEQ ID NO.100 to SEQ ID NO.132, and the mutation site of the SNP is located at position 201 of the sequence of SEQ ID NO.100 to SEQ ID NO.132;

[0022] (3) Construct a fingerprint map of thin-shelled pecan based on the test results.

[0023] Optionally, in step 2), the genotype of the SNP can be detected by sequencing or KASP or any other available genotyping technology.

[0024] On the other hand, another object of the present invention is to provide a method for identifying or assisting in identifying a thin-shell pecan variety, characterized in that it comprises:

[0025] (1) extracting total DNA of the thin-shelled walnut sample to be identified;

[0026] (2) detecting the genotype of each SNP molecular marker in the aforementioned fingerprint spectrum in the sample;

[0027] (3) Determine the variety of the thin-shell pecan sample to be identified based on the test results.

[0028] 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.

[0029] The specific judgment method of step 3) is that when the genotype of the thin-shell pecan sample to be identified is consistent with the genotype of any variety in the aforementioned fingerprint map, it is judged to be the corresponding thin-shell pecan variety.

[0030] In another aspect, the present invention provides any of the following applications of the aforementioned method:

[0031] (1) Application in identification of thin-shell pecan varieties;

[0032] (2) Application in identification, improvement or molecular marker-assisted breeding of walnut germplasm resources;

[0033] (3) Application in screening or creating different thin-shell pecan varieties;

[0034] (4) Application in constructing a DNA fingerprint database of thin-shelled pecan.

[0035] The present invention has the following beneficial effects: a new SNP molecular marker is developed, and these SNP sites can form a fingerprint map for the identification of 112 species of thin-shelled pecans, which provides a guarantee for the identification, resource utilization and breeding of thin-shelled pecans, and is also of great significance for the molecular marker breeding of thin-shelled pecans. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] 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.

[0037] Figure 1 It is a fingerprint map drawn from the KASP test results of 114 samples to be tested. DETAILED DESCRIPTION

[0038] 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.

[0039] 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.

[0040] Example 1 Resequencing Analysis

[0041] 1. Experimental Materials

[0042] The source of materials and information are shown in Table 2:

[0043] Table 2 Sources and information

[0044] 2. Sample DNA extraction and library construction and sequencing

[0045] 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.

[0046] Specific experimental steps:

[0047] The library was constructed with 1 μg DNA as the starting amount;

[0048] High-quality genomic DNA was extracted using the CTAB method;

[0049] 0.75% agarose gel electrophoresis was used to detect the size of DNA fragments and the degree of DNA degradation;

[0050] 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;

[0051] 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.

[0052] 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;

[0053] The qualified libraries were arranged to be loaded onto the machine for 2*150bp sequencing.

[0054] 3. Data quality control

[0055] (1) Remove the adapter sequence from the sequence;

[0056] (2) Remove polyG and polyX at the end of the reads (minimum length is 10 bp);

[0057] (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;

[0058] (4) Remove reads with N greater than 5;

[0059] (5) Remove reads with bases with a quality lower than 15 accounting for more than 40%;

[0060] (6) Remove reads with a length of less than 15 bp after filtering.

[0061] 4. Data comparison

[0062] 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.

[0063] 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:

[0064] (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;

[0065] (2) Use samtools (parameter: sort) to convert the alignment results from sam (Sequence Alignment / MAP) files to sorted bam files (binary Alignment / Map);

[0066] (3) Use samtools (parameter: markdup -r) to remove duplicates from the sorted alignment results for subsequent analysis;

[0067] (4) Use Python scripts to calculate the alignment rate and coverage.

[0068] 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%).

[0069] A total of 31 365 448 SNP loci were developed.

[0070] Example 2 Development of core SNPs for identification of 112 pecan varieties

[0071] 1. Identification of core SNPs

[0072] According to the grouping of 112 thin-shell pecans, the SNP combination that distinguishes 112 thin-shell pecan varieties was selected. At the same time, in order to reduce the false positive of SNP, the following analysis criteria were adopted:

[0073] 1. Site detection rate = 100%; 2. MAF greater than 0.15; 3. Heterozygosity rate less than 0.4; 4. Redundant markers removed;

[0074] 5. There are no other SNPs within 30bp before and after the selected SNP site; 6. GC content: Analyze the GC content of the SNP site to avoid selecting sites with too high or too low GC content. The GC content of 150bp before and after the selected SNP site is 40%-60%; 7. PIC >= 0.2; 8. Site polymorphism >= 0.4; 9. There cannot be >= 8 consecutive single base repeats within 30bp upstream and downstream of the SNP site; 10. There cannot be homology between the 50bp upstream and downstream of the SNP site and other positions in the genome.

[0075] Finally, 33 SNPs were screened. The genotypes of the 33 core SNP loci in different varieties were sorted from small to large according to chromosome number and position as shown in Table 3. The specific sorting order is CHR1_6071055, CHR1_42893205, CHR1_47954051, CHR1_52228116, CHR1_57469932, CHR2_26663233, CHR3_2564907, CHR3_3638372, CHR3_7034444, CHR3_52781451, CHR4_5937447, CHR4_28328637, CHR5_2793044, CHR5_10451065, CHR7_35325592, CHR8_8984149 、CHR9_2652188、CHR10_6827793、CHR11_4001936、CHR11_5157026、CHR11_45224077、CHR12_19588842、CHR12_24676837、CHR13_694161、CHR13_2422699、CHR13_19946268、CHR13_25358204、CHR14_11071395、CHR14_17739311、CHR14_19889290、CHR15_19954498、CHR15_37055200、CHR16_3365513, the genotype combination of these different SNPs constitutes the fingerprint map.

[0076] Table 3 Multiple sequence alignment results of the 112 sample SNP sites arranged in ascending order of SNP numbers

[0077]

[0078] Example 3 Molecular marker verification

[0079] 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 species samples No. 113-114);

[0080] 2) Primer and probe design;

[0081] 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 4.

[0082] Table 4 kasp primers

[0083]

[0084] 3) Using the primers and probes designed in step 2) to perform KASP gene detection typing on the qualified DNA extracted in step 1). The primers and probes are combined to prepare a detection kit.

[0085] 4) Determine the type of sample based on the test results.

[0086] Results Figure 1 , Figure 1 The fingerprint map showed that the genotypes of 112 varieties were completely different, which was consistent with the resequencing results and was also completely different from the genotypes of samples 113-114.

[0087] In summary, the fingerprint prepared by the present invention can be used for the accurate identification of 112 varieties.

[0088] 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 KASP primer set for detecting SNP molecular markers of Carya syringae or constructing a fingerprint of Carya syringae, characterized in that: The primer set consists of the following 33 primers: Primer set 1: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3; Primer set 2: SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6; Primer set 3: SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9; Primer set 4: SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12; Primer set 5: SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15; Primer set 6: SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18; Primer set 7: SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21; Primer set 8: SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24; Primer set 9: SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27; Primer set 10: SEQ ID NO.28, SEQ ID NO.29, SEQ ID NO.30; Primer set 11: SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.33; Primer set 12: SEQ ID NO.34, SEQ ID NO.35, SEQ ID NO.36; Primer set 13: SEQ ID NO.37, SEQ ID NO.38, SEQ ID NO.39; Primer set 14: SEQ ID NO.40, SEQ ID NO.41, SEQ ID NO.42; Primer set 15: SEQ ID NO.43, SEQ ID NO.44, SEQ ID NO.45; Primer set 16: SEQ ID NO.46, SEQ ID NO.47, SEQ ID NO.48; Primer set 17: SEQ ID NO.49, SEQ ID NO.50, SEQ ID NO.51; Primer set 18: SEQ ID NO.52, SEQ ID NO.53, SEQ ID NO.54; Primer set 19: SEQ ID NO.55, SEQ ID NO.56, SEQ ID NO.57; Primer set 20: SEQ ID NO.58, SEQ ID NO.59, SEQ ID NO.60; Primer set 21: SEQ ID NO.61, SEQ ID NO.62, SEQ ID NO.63; Primer set 22: SEQ ID NO.64, SEQ ID NO.65, SEQ ID NO.66; Primer set 23: SEQ ID NO.67, SEQ ID NO.68, SEQ ID NO.69; Primer set 24: SEQ ID NO.70, SEQ ID NO.71, SEQ ID NO.72; Primer set 25: SEQ ID NO.73, SEQ ID NO.74, SEQ ID NO.75; Primer set 26: SEQ ID NO.76, SEQ ID NO.77, SEQ ID NO.78; Primer set 27: SEQ ID NO.79, SEQ ID NO.80, SEQ ID NO.81; Primer set 28: SEQ ID NO.82, SEQ ID NO.83, SEQ ID NO.84; Primer set 29: SEQ ID NO.85, SEQ ID NO.86, SEQ ID NO.87; Primer set 30: SEQ ID NO.88, SEQ ID NO.89, SEQ ID NO.90; Primer set 31: SEQ ID NO.91, SEQ ID NO.92, SEQ ID NO.93; Primer set 32: SEQ ID NO.94, SEQ ID NO.95, SEQ ID NO.96; Primer set 33: SEQ ID NO.97, SEQ ID NO.98, SEQ ID NO.99, the sequence of the SNP molecular marker is shown in SEQ ID NO.100~SEQ ID NO.132, the mutation site of the SNP is located at the 201st position of the SEQ ID NO.100~SEQID NO.132 sequence, and the fingerprint spectrum is composed of the SNP molecular marker.

2. A kit for identifying different varieties of thin-shelled pecans, characterized in that: The kit comprises the primer set according to claim 1.

3. Any of the following uses of the primer set according to claim 1 or the kit according to claim 2: (1) Application in identification of thin-shell pecan varieties; (2) Application in constructing the DNA fingerprint database of thin-shelled pecan.

4. A method for constructing a fingerprint of thin-shelled walnuts, characterized in that: The method comprises the following steps: (1) Extracting total DNA from samples of different varieties of thin-shelled pecans; (2) Detecting the genotype of the SNP molecular marker in the thin-shelled walnut sample, the sequence of the SNP molecular marker is shown in SEQ ID NO. 100 to SEQ ID NO. 132, and the mutation site of the SNP is located at the 201st position of the sequence of SEQ ID NO. 100 to SEQ ID NO. 132; (3) Construct a fingerprint map of thin-shelled pecan based on the test results.

5. The method according to claim 4, characterized in that Step (2) Detecting the genotype of the SNP molecular marker by sequencing or KASP or any other available genotyping technology.

6. A method for identifying a thin-shell pecan variety, characterized in that: The method comprises the following steps: (1) extracting total DNA of the thin-shelled walnut sample to be identified; (2) Detecting the genotype of the SNP molecular marker in the thin-shelled walnut sample, the sequence of the SNP molecular marker is shown in SEQ ID NO. 100 to SEQ ID NO. 132, and the mutation site of the SNP is located at the 201st position of the sequence of SEQ ID NO. 100 to SEQ ID NO. 132; (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 by sequencing or KASP or any other available genotyping technology.

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