SNP molecular marker for identifying carya illinoinensis variety 'zhongxiu 15' and application thereof

Abstract

The present application relates to the technical field of molecular markers, and particularly relates to a SNP molecular marker of 'Zhongxiu 15' Carya illinoinensis and application thereof. The SNP molecular marker can be used to identify 'Zhongxiu 15' Carya illinoinensis, and is of great significance to germplasm resource identification of Carya illinoinensis and molecular marker breeding.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the identification of SNP molecular markers for the thin-shelled pecan variety 'Zhongxiu 15' and their applications. Background Technology

[0002] Pecan (Carya illinoinensis), a deciduous tree belonging to the genus Carya in the family Juglandaceae, is also known as the American pecan and is native to the Americas. The fruit is commercially known as pecan. Its kernels are rich in unsaturated fatty acids such as oleic and linoleic acid, as well as protein, polyphenols, flavonoids, and trace elements, offering benefits such as brain health, anti-aging, and prevention of cardiovascular diseases; hence, it is also called the "longevity fruit." In recent years, with breakthroughs in breeding technology, the planting area of ​​pecans has been continuously expanding. However, the booming seedling market has also brought a series of problems, such as numerous instances of "different names for the same species" and "different species with the same name"; the substitution of inferior or even other species of seedlings for superior ones also occurs frequently.

[0003] 'Zhongxiu 15' is a seedling variety. The nut is oblong, with pointed ends, and the shell surface has brownish-black to black striped markings with few spots. The average nut weight is 5.11 g, with a longitudinal diameter of 41.88 mm, a transverse diameter of 18.94 mm, and a fruit shape index of 2.21. The average oil content of the kernel is 60.17%. The kernel is golden yellow and very plump; the shell is of medium thickness, about 0.85 mm, and easy to remove. 'Zhongxiu 15' has large fruits and a high oil yield, which are significant advantages compared to other varieties. Although these characteristics are prominent, it is difficult to identify 'Zhongxiu 15' through phenotypic analysis during the seedling stage before fruiting. Combining molecular biology and phenotypic data will yield more convincing conclusions. Therefore, DNA barcoding based on molecular marker technology, with its high throughput and sensitivity, can be used for large-scale and accurate identification of 'Zhongxiu 15'. Summary of the Invention

[0004] This invention addresses the aforementioned technical problems by providing a simple molecular marker method for the identification of 'Zhongxiu 15' thin-shelled pecan. Specifically, this invention provides the following technical solution:

[0005] In a first aspect, the present invention provides a DNA barcode for identifying or assisting in the identification of the 'Zhongxiu 15' thin-shelled pecan variety, characterized in that the sequence of the DNA barcode is CGAGCWAG, and the positions of the bases of the DNA barcode on the chromosome are as shown in the following table:

[0006] .

[0007] This invention utilizes high-throughput sequencing to identify eight SNP loci within the whole genome of the thin-shelled pecan variety 'Zhongxiu 15'. These SNP loci can form a unique DNA barcode for 'Zhongxiu 15'. Those skilled in the art will understand that the DNA barcode described in this invention is not composed of continuous bases, but rather of genotypes of SNPs dispersed at different loci on different chromosomes. Therefore, the DNA barcode described in this invention can also be referred to as a genotype barcode.

[0008] Furthermore, the sequences of the SNP sites in the aforementioned DNA barcode are shown in SEQ ID NO.25~SEQ ID NO.32 (in order of SNP position in the DNA barcode):

[0009] .

[0010] In particular, the genotype of locus CHR13_25358204 (chromosome 13, locus 25358204) is AA, which is different from all other varieties. Since the propagation of pecan varieties after breeding is through single-plant asexual reproduction to ensure the stability of varietal traits, the AA genotype at locus CHR13_25358204 can be considered a unique locus for 'Zhongxiu 15' and can be used for the identification or auxiliary identification of this variety.

[0011] Furthermore, the sequence of the SNP molecular marker CHR13_25358204 is shown in SEQ ID NO. 31. The SNP molecular marker CHR13_25358204 is an A / G mutation located at position 201 of SEQ ID NO. 31. The genotype of the SNP molecular marker CHR13_25358204 in the 'Zhongxiu 15' thin-shelled pecan variety is AA.

[0012] On the other hand, the present invention provides KASP primers for detecting the aforementioned SNP molecular markers, the primer sequences of which are shown in SEQ ID NO.1~24.

[0013] .

[0014] On the other hand, the present invention provides a kit for identifying or assisting in the identification of the 'Zhongxiu 15' thin-shelled pecan 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 barcodes, molecular markers, primers, and kits:

[0016] (1) Application in identifying the 'Zhongxiu 15' thin-shelled hickory variety;

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

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

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

[0020] On the other hand, another object of the present invention is to provide a method for identifying or assisting in the identification of the 'Zhongxiu 15' thin-shelled pecan variety, characterized in that it includes:

[0021] (1) Extract total DNA from the thin-shelled pecan samples to be identified;

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

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

[0024] Optionally, step 2) can be performed by sequencing or KASP or any other available genotyping technique to detect the genotype of each base of the aforementioned DNA barcode.

[0025] The specific judgment method for step 3) is as follows: if the genotype of the thin-shelled pecan sample to be identified is inconsistent with the aforementioned DNA barcode, it is judged to be a non-'Zhongxiu 15' thin-shelled pecan variety.

[0026] Furthermore, in step 3), if the genotype of the thin-shelled pecan sample to be identified matches the aforementioned DNA barcode, it is determined to be the 'Zhongxiu 15' thin-shelled pecan variety.

[0027] On the other hand, another object of the present invention is to provide another method for identifying or assisting in the identification of the 'Zhongxiu 15' thin-shelled pecan variety, characterized by comprising:

[0028] (1) Extract total DNA from the thin-shelled pecan samples to be identified;

[0029] (2) Detect the genotype of the SNP molecular marker CHR13_25358204;

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

[0031] Optionally, step 2) can be performed by sequencing or KASP or any other available genotyping technique to detect the genotype of the aforementioned SNP molecular marker.

[0032] The specific judgment method for step 3) is as follows: if the genotype of the thin-shelled pecan sample to be identified is inconsistent with the genotype of the aforementioned SNP molecular marker CHR13_25358204, it is determined that it is not the 'Zhongxiu 15' thin-shelled pecan variety.

[0033] Furthermore, in step 3), if the genotype of the thin-shelled pecan sample to be identified matches the genotype of the SNP molecular marker CHR13_25358204, it is determined to be the 'Zhongxiu 15' thin-shelled pecan variety.

[0034] The aforementioned identification or auxiliary identification means directly identifying the sample to be tested as the 'Zhongxiu 15' thin-shelled hickory variety, or using it only as an auxiliary means to exclude the sample to be tested from being the 'Zhongxiu 15' thin-shelled hickory variety.

[0035] Furthermore, the aforementioned identification or auxiliary identification methods can at least distinguish 'Zhongxiu 15' from any other variety described in this invention.

[0036] On the other hand, the present invention provides any of the following applications of the aforementioned method:

[0037] (1) Application in identifying the 'Zhongxiu 15' thin-shelled hickory variety;

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

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

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

[0041] This invention offers the following advantages: by using a single DNA barcode or SNP molecular marker, it is possible to effectively distinguish whether the tested variety is 'Zhongxiu 15' thin-shelled pecan, thus providing a guarantee for the identification, planting, resource utilization, and breeding of 'Zhongxiu 15' thin-shelled pecan. It also has significant implications for molecular marker breeding of thin-shelled pecans. Attached Figure Description

[0042] The method of the present invention and its beneficial effects will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0043] Figure 1 These are the test results for 114 samples.

[0044] Figure 2 This is the genotyping result of CHR13_25358204 loci from 114 samples. The samples are arranged in order of number from left to right starting from the first row (the last black square has no sample); Figure A shows samples 1 to 94, and Figure B shows samples 95 to 114. Detailed Implementation

[0045] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort 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 meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The pecan plant samples used in the following embodiments are commercially available or collected from the wild.

[0047] Example 1 Resequencing Analysis

[0048] 1. Experimental Materials

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

[0050] Table 1. Material Sources and Information (Notes indicate different names for the same sample)

[0051]

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

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

[0054] Specific experimental steps:

[0055] Library construction was initiated with 1 μg of DNA;

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

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

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

[0059] The Qubit 3.0 fluorescence analyzer (Life Technologies, Carlsbad, CA, USA) was used to detect DNA concentrations greater than 50 ng / μl and total amounts greater than 2 μg.

[0060] After DNA was broken down by sonication with a Covaris M220, magnetic beads were used for fragment selection, resulting in sample bands concentrated between 200-400 bp.

[0061] The qualified libraries were then put into the sequencing 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 from the end of the reads (the shortest length is 10bp);

[0065] (3) The average quality value of the bases in the window is calculated by using a sliding window method. Low-quality sliding windows are clipped out. Its function is similar to Trimmomatic.

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

[0067] (5) Remove reads with a base content of less than 15 that is higher than 40%;

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

[0069] 4. Data comparison

[0070] In this invention, we use the genome of thin-shelled pecan as a reference genome, use BWA alignment software to align the sequencing fragments back to the reference genome, and then use Picard-tools to remove the sequencing fragments generated by PCR-duplication.

[0071] For each sample, a bwa (version: 0.7.17; parameter: mem) alignment analysis was performed. The filtered clean reads were then aligned to the reference genome, and the alignment results were statistically analyzed. The specific analysis steps are as follows:

[0072] (1) Use bwa alignment software (parameter: mem -R, other parameters are the 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 the sam (Sequence Alignment / MAP) file to a sorted bam (binary Alignment / Map) file;

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

[0075] (4) Statistical comparison rate and coverage.

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

[0077] A total of 31,365,448 SNP sites were discovered.

[0078] Example 2: Development of core SNPs for identifying 'Jong-soo 15'

[0079] 1. Identification of core SNPs

[0080] Based on the grouping of 'Zhongxiu 15' and other thin-shelled pecan varieties, SNPs were selected to distinguish 'Zhongxiu 15' from other thin-shelled pecan varieties. The selection criteria were that no more than 50% of the loci shared the same genotype as 'Zhongxiu 15', and that the combined SNPs could identify 'Zhongxiu 15'. To reduce false positives, the following analytical criteria were adopted:

[0081] 1. No other SNPs are found within 30 bp upstream and downstream of the snp site;

[0082] 2. There must be no homology within 50 bp upstream and downstream of the SNP site and at other locations in the genome;

[0083] 3. There cannot be more than 8 consecutive single base repeats within 30 bp upstream and downstream of the SNP site;

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

[0085] 5. Extract the remaining 250bp upstream and downstream sequences from the markers to design primers. Product size: 250-400. Align the designed primers with the genome for homology and filter the primers to match primers from multiple locations.

[0086] Finally, eight markers were selected as core SNPs, and these eight core SNP loci were used as the core SNP loci for identifying 'Zhongxiu 15'. The genotypes of the eight core SNP loci in different varieties were sorted from smallest to largest according to chromosome number and position, as shown in Table 2. The specific sorting order is CHR1_42893205, CHR1_57469932, CHR3_2564907, CHR3_3638372, CHR5_10451065, CHR12_19588842, CHR13_25358204, and CHR14_11071395.

[0087] The genotype combinations at these eight loci in 'Zhongxiu 15' form the barcode sequence CGAGCWAG, which is completely different from the SNP genotype combinations of other varieties (Table 2), and can be used as a DNA barcode for identifying 'Zhongxiu 15'. In particular, the genotype of locus CHR13_25358204 is AA, which is different from all other varieties. Considering that the propagation of pecan varieties after breeding is done through single-plant asexual reproduction to ensure the stability of varietal traits, the AA genotype at locus CHR13_25358204 can be considered a unique locus of 'Zhongxiu 15' and can be used for the identification of this variety.

[0088]

[0089] Example 3 Molecular marker verification

[0090] 1) Extract total DNA from the samples to be tested (independently verify that the sample variety number is consistent with Example 1, and add non-thin-shelled pecan species samples 113-114).

[0091] 2) Primer and probe design;

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

[0093]

[0094] 3) Using the primers and probes designed in step 2), the qualified DNA extracted in step 1) is subjected to genotyping using KASP detection. The primers and probes are then combined to prepare a detection kit.

[0095] 4) Determine the variety of the sample based on the test results.

[0096] See results Figures 1-2 , Figure 1 The sample numbered 67 is the 'Zhongxiu 15' variety, and its genotype composition sequence at 8 loci is CGAGCWAG, which is completely different from the genotypes of other samples. Figure 2 The results showed that the CHR13_25358204 site of the 'Zhongxiu 15' variety was AA, which was completely different from other samples.

[0097] In summary, the DNA barcode prepared by this invention can be used for the accurate identification of the 'Zhongxiu 15' variety, and the single SNP site CHR13_25358204 can also directly identify the 'Zhongxiu 15' variety.

[0098] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to the above embodiments will be readily apparent to those 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A SNP molecular marker CHR13_25358204 for identifying the 'Zhongxiu 15' thin-shelled pecan variety, characterized in that, The sequence of the SNP molecular marker CHR13_25358204 is shown in SEQ ID NO.

31. The SNP molecular marker CHR13_25358204 is a G / A mutation located at position 201 of SEQ ID NO.

31. The genotype of the SNP molecular marker CHR13_25358204 in the 'Zhongxiu 15' thin-shelled pecan variety is AA.

2. A SNP molecular marker for identifying the 'Zhongxiu 15' thin-shelled pecan variety, characterized in that... , The sequences of the SNP molecular markers are shown in SEQ ID NO.25~SEQ ID NO.32, and the SNP molecular markers are listed in the following table in order: 。 3. A KASP primer for detecting the SNP molecular marker of claim 1 or claim 2, characterized in that, The KASP primers are used to detect the sequences of the KASP primers for the SNP molecular markers of claim 1 as shown in SEQ ID NO.19~SEQ ID NO.21, and to detect the sequences of the SNP molecular markers of claim 2 as shown in SEQ ID NO.1~SEQ ID NO.

24.

4. A kit for identifying the 'Zhongxiu 15' thin-shelled pecan variety, the kit comprising the primers as described in claim 3.

5. Any of the following applications of the SNP molecular marker of claim 1, the SNP molecular marker of claim 2, the primer of claim 3, or the kit of claim 4: (1) Application in identifying the 'Zhongxiu 15' thin-shelled hickory variety; (2) Application in the identification, improvement or molecular marker-assisted breeding of thin-shelled pecan germplasm resources; (3) Application in constructing a DNA fingerprint database for thin-shelled pecans.

6. A method for identifying the 'Zhongxiu 15' thin-shelled hickory variety, characterized in that, include: (1) Extract total DNA from the thin-shelled pecan samples to be identified; (2) Detect the genotype of the SNP molecular marker CHR13_25358204 as described in claim 1; (3) Determine the variety of the thin-shelled pecan sample to be identified based on the test results.

7. The method as described in claim 6, characterized in that, Step 2) The genotype of the molecular marker CHR13_25358204 described in claim 1 can be detected by sequencing, KASP, or any other available genotyping technique.

8. A method for identifying the 'Zhongxiu 15' thin-shelled hickory variety, characterized in that, include: (1) Extract total DNA from the thin-shelled pecan samples to be identified; (2) Detect the genotype of the SNP molecular marker described in claim 2; (3) Determine the variety of the thin-shelled pecan sample to be identified based on the test results.

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

10. Any of the following applications of the method according to any one of claims 6 to 9: (1) Application in identifying the 'Zhongxiu 15' thin-shelled hickory variety; (2) Application in the identification, improvement or molecular marker-assisted breeding of thin-shelled pecan germplasm resources; (3) Application in constructing a DNA fingerprint database for thin-shelled pecans.

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