SNP molecular marker for identifying carya illinoensis variety yaogou 3 and application thereof

Abstract

The present application relates to the technical field of molecular marker, in particular to a SNP molecular marker of 'Yaogou No.3' Carya illinoinensis and application thereof. The SNP molecular marker of the present application can be used to identify 'Yaogou No.3' Carya illinoinensis, and has important significance for germplasm resource identification and molecular marker breeding of Carya illinoinensis.

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-shelled walnut variety 'Yaogou No. 3' and an application thereof. Background Art

[0002] The thin-shelled pecan (Carya illinoinensis), a large deciduous tree of the genus Carya in the Juglandaceae family, is also known as the American pecan and is native to the Americas. Its fruit is delicious, non-astringent, and it produces high yields. Its straight trunk and high-quality wood make it a world-renowned ecological and economical tree. The kernel, rich in unsaturated fatty acids such as oleic and linoleic acids, as well as protein, polyphenols, flavonoids, and trace elements, boasts benefits such as brain-boosting, anti-aging, and cardiovascular disease prevention. It is also known as the longevity fruit and is deeply loved by the public. In recent years, breakthroughs in breeding technology have led to a continuous expansion in planted areas. However, instances of "same name for same thing" and "same name for different thing" remain numerous. Substandard products, or even seedlings from other species, are also used as substitutes. Therefore, identifying and verifying seedling varieties is a pressing issue in the promotion and application of thin-shelled pecan seedlings.

[0003] Yaogou No. 3, a single seedling, is a new and superior variety bred by the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province. Its fruit matures in mid-October. The nut is oblong, with an asymmetrical, acute-angled apex and a rounded base. The shell is spotted with brownish-black to black stripes and numerous spots. The average nut weight is 8.41 g, with a longitudinal diameter of 39.98 mm and a transverse diameter of 23.00 mm, and a fruit shape index of 1.842. The average kernel content is 55.67%, and the kernel oil content is 73.82% (the highest oil content under the same measurement conditions). The kernel contains 376.47 mg / g of unsaturated fatty acids, accounting for 93.16% of the total fatty acid content, of which monounsaturated fatty acids account for 72.60% (the kernel has a high monounsaturated fatty acid content), and polyunsaturated fatty acids account for 27.40%. The kernel is golden yellow and very plump. The shell is medium-thick, approximately 0.748 mm thick, making it easy to shell. Although Yaogou No. 3 offers numerous advantages and differences compared to other thin-shelled pecan varieties, efficient identification remains challenging for the average technician. Furthermore, phenotypic data alone is subjective, requiring the integration of molecular biological evidence (e.g., fingerprinting) to provide a more convincing diagnosis. While there are currently available molecular markers for pecans, most are limited to AFLPs and SSRs. Furthermore, no molecular markers specifically designed for Yaogou No. 3 have been reported. DNA barcodes, developed based on molecular marker technology, offer high throughput and sensitivity, enabling accurate, large-scale identification of Yaogou No. 3. Summary of the Invention

[0004] The present invention primarily addresses the above technical issues and provides a simple molecular marker method for identifying the 'Yaogou No. 3' thin-shelled pecan. 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 utilizes high-throughput sequencing to identify seven single-nucleotide polymorphisms (SNPs) within the entire genome of the 'Yaogou No. 3' var. calcarya. These SNPs constitute a unique DNA barcode for 'Yaogou No. 3'. Those skilled in the art will appreciate that the DNA barcode described herein is not composed of continuous bases but rather consists of genotypes dispersed across different chromosomes and loci. Therefore, the DNA barcode described herein can also be referred to as a genotypic barcode.

[0008] Furthermore, the sequences of the SNP sites in the above DNA barcodes are shown as 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 at locus CHR5_2793044 (chromosome 5, locus 2793044) is GG, which is different from all other varieties. Since pecan varieties are propagated through single-plant asexual propagation to ensure the stability of their traits, the GG genotype at locus CHR5_2793044 can be used as a unique locus for 'Yaogou No. 3' and can be used for identification or auxiliary identification of this 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. The genotype of the SNP molecular marker CHR5_2793044 in the 'Yaogou No. 3' thin-shelled 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 to 21.

[0013] .

[0014] In another aspect, 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 barcodes, molecular markers, primers, and kits:

[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-shelled pecan varieties;

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

[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-shelled pecan variety, characterized by comprising:

[0021] (1) extracting the 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-shelled 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-shelled walnut sample to be identified is inconsistent with the aforementioned DNA barcode, it is judged to be not the 'Yaogou No. 3' thin-shelled walnut variety.

[0026] Furthermore, in 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-shelled pecan variety, characterized by comprising:

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

[0029] (2) Detect the genotype of the SNP molecular marker CHR5_2793044;

[0030] (3) Determine the variety of the thin-shelled 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-shelled pecan 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-shelled pecan 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 CHR5_2793044, it is determined to be the 'Yaogou No. 3' thin-shelled walnut variety.

[0034] The aforementioned identification or auxiliary identification means directly determining that the sample to be tested is the 'Yaogou No. 3' thin-shelled pecan variety, or only serving as an auxiliary means to exclude that the sample to be identified is the 'Yaogou No. 3' thin-shelled pecan variety.

[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-shelled pecan varieties;

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

[0041] The present invention has the following beneficial effects: using a single DNA barcode or SNP molecular marker, it can effectively distinguish whether the tested variety is the 'Yaogou No. 3' thin-shelled pecan, providing assurance for the identification, planting, resource utilization, and breeding of the 'Yaogou No. 3' thin-shelled pecan. It is also of great significance for molecular marker breeding of thin-shelled pecans. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0044] Figure 2 Figure 1 shows the typing results of the CHR5_2793044 locus for 114 samples. The samples are sorted by number starting from the first row and placed from left to right (the black square at the end has no sample). Figure A shows samples 1 to 94, and Figure B shows samples 95 to 114. DETAILED DESCRIPTION

[0045] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. 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 making any creative efforts 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 skilled 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 of DNA as the starting amount;

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

[0057] DNA fragment size and DNA degradation degree were detected by 0.75% agarose gel electrophoresis;

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

[0059] The DNA concentration was detected using a 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 μg.

[0060] After 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 loaded onto the machine for 2*150bp sequencing.

[0062] 3. Data quality control

[0063] (1) Eliminate the adapter sequence in the sequence;

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

[0065] (3) Calculate the average quality value of the bases in the window in a sliding window manner, and trim the sliding window with low quality. Its function is similar to Trimmomatic;

[0066] (4) Eliminate 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] Each sample was analyzed using bwa (version: 0.7.17; parameters: mem). The filtered clean reads were aligned to the reference genome and the alignment statistics were calculated. 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) Statistical comparison rate and coverage.

[0076] Using 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 identifying 'Yaogou No. 3'

[0079] 1. Identification of core SNPs

[0080] Based on the grouping between 'Yaogou No. 3' and other thin-shelled pecans, SNPs were selected to distinguish 'Yaogou No. 3' from other thin-shelled pecans. The selection criteria were that no more than 50% of the loci had the same genotype as 'Yaogou No. 3' and that the combination could identify 'Yaogou No. 3'. To reduce false positives for SNPs, the following analysis criteria were used:

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

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

[0085] 5. Extract the remaining markers and design primers based on the 250bp upstream and downstream sequences. The product size is 250-400. The designed primers are homologously aligned with the genome, and primers that can align to multiple locations are filtered.

[0086] The final seven markers were selected as core SNPs and used as the core SNP loci for identifying Yaogou No. 3. The genotypes of the seven core SNP loci in different varieties were sorted by chromosome number and position from smallest to largest, as shown in Table 2. The specific sorting order is CHR2_26663233, CHR3_2564907, CHR4_5937447, CHR5_2793044, CHR5_10451065, CHR11_4001936, and CHR12_24676837.

[0087] The genotype combination of these seven loci in 'Yaogou No. 3' is sequenced and combined into a barcode sequence of RAMGCCW. The genotypes of these seven loci are completely different from the SNP genotype combinations of other varieties (Table 2), making them suitable for use as DNA barcodes for identifying 'Yaogou No. 3'. In particular, the genotype of locus CHR5_2793044, GG, is distinct from all other varieties. Considering that pecan varieties are propagated through single-plant asexual propagation to ensure stable varietal traits, the GG genotype at locus CHR5_2793044 can be considered unique to 'Yaogou No. 3' and can be used for identification of this variety.

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

[0089]

[0090] Example 3 Molecular marker verification

[0091] 1) Extract total DNA from the sample to be tested (independently verify that the sample species 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), perform KASP detection and genotyping on the qualified DNA extracted in step 1). The primers and probes are combined to form a detection kit.

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

[0098] See the results Figures 1 and 2 , Figure 1 The results showed that sample No. 47 was 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 'Yaogou No. 3' was GG, which was completely different from 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 is intended to enable one skilled in the art to implement or use the present invention. Various modifications to the above embodiments will be readily 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 is not limited to the embodiments shown herein but is to be construed in the widest possible manner consistent with the principles and novel features disclosed herein.

Claims

1. A SNP molecular marker CHR5_2793044 for identifying the 'Yaogou No. 3' thin-shelled walnut variety, characterized by: The nucleotide 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 of SEQ ID NO.

25. The genotype of the SNP molecular marker CHR5_2793044 in the 'Yaogou No. 3' thin-shelled walnut variety is GG.

2. A SNP molecular marker combination for identifying the 'Yaogou No. 3' thin-shelled walnut variety, characterized in that: The nucleotide sequences of the SNP molecular marker combination are shown in SEQ ID NO.22 to SEQ ID NO.28, and the mutation types are shown in the following table: 。 3. The KASP primer for detecting the SNP molecular marker CHR5_2793044 according to claim 1, characterized in that: The nucleotide sequences of the primers are shown in SEQ ID NO.10 to SEQ ID NO.

12.

4. The KASP primers for detecting the SNP molecular marker combination according to claim 2, characterized in that: The nucleotide sequences of the primers are shown in SEQ ID NO.1 to SEQ ID NO.

21.

5. A kit for identifying the 'Yaogou No. 3' thin-shelled walnut variety, comprising the primers according to claim 3 or 4.

6. Use of the SNP molecular marker CHR5_2793044 according to claim 1, the molecular marker combination according to claim 2, the primer according to claim 3 or 4, or the kit according to claim 5 in identifying the 'Yaogou No. 3' thin-shelled walnut variety.

7. A method for identifying the 'Yaogou No. 3' thin-shelled walnut variety, characterized in that: include: (1) extracting the total DNA of the thin-shelled walnut sample to be identified; (2) detecting the genotype of the SNP molecular marker CHR5_2793044 described in claim 1; (3) Determine the variety of the thin-shelled pecan sample to be identified based on the test results.

8. The method according to claim 7, wherein Step 2) detecting the genotype of the SNP molecular marker CHR5_2793044 of claim 1 by sequencing or KASP genotyping technology.

9. A method for identifying the 'Yaogou No. 3' thin-shelled walnut variety, characterized in that: include: (1) extracting the total DNA of the thin-shelled walnut sample to be identified; (2) detecting the genotype of the SNP molecular marker combination described in claim 2; (3) Determine the variety of the thin-shelled pecan sample to be identified based on the test results.

10. The method according to claim 9, wherein Step 2) detecting the genotype of the SNP molecular marker combination according to claim 2 by sequencing or KASP genotyping technology.

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