KASP Primers for Identifying Different Carya illinoinensis Varieties and Their Applications

Abstract

The present invention relates to the technical field of molecular markers, and particularly relates to KASP primers, SNP molecular markers, fingerprint maps of 112 pecan varieties and their applications. By adopting the technical scheme of the present invention, a fingerprint map of pecan can be drawn, which is used for accurate identification of pecan varieties and is of great significance for the identification of germplasm resources of pecan and molecular marker breeding.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to KASP primers, SNP molecular markers, fingerprint maps for identifying different pecan varieties and their applications. Background Art

[0002] Pecan (Carya illinoinensis (Wangenh.) K.Koch) is a deciduous tree of the genus Carya in the Juglandaceae family. Pecan kernels are 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, and have the effects of enhancing brainpower, delaying aging, preventing cardiovascular and cerebrovascular diseases, etc.

[0003] In recent years, with the breakthrough of breeding techniques, the seedling sales market has been continuously heating up. However, the situations of "different names for the same species" and "the same name for different species" in the market are numerous; the phenomena of passing off inferior goods as good ones, or even using seedlings of other species to "make up the number" also occur from time to time.

[0004] Kompetitive allele specific PCR (KASP) is a new genotyping technology based on single nucleotide polymorphism (SNP), which can accurately genotype SNPs and insertion-deletion polymorphisms (Indels) at the genomic level. Through the KASP genotyping technology, high-throughput, low-cost, rapid and simple verification can be carried out, and the screened markers can be applied to variety identification and discrimination, as well as molecular marker-assisted breeding. The KASP technology has been widely applied in molecular marker-assisted selection breeding of various crops, but it has not been reported in pecan. Although there are some molecular markers for pecan in the prior art, most of them are limited to AFLPs, SSRs, etc. Collecting and sorting out the core germplasm resources of pecan to develop new core SNP molecular markers that can be used for pecan germplasm identification and constructing fingerprint maps can establish variety tags for a large population of pecan. Summary of the Invention

[0005] The present invention mainly aims at the above technical problems, provides a simple method for molecular markers, constructs a fingerprint map of pecan, and realizes the identification of 112 different pecan varieties. Specifically, the present invention provides the following technical solutions:

[0006] In the first aspect, the present invention provides a combination of SNP molecular markers for identifying or assisting in identifying pecan varieties, characterized in that the positions of the SNP molecular markers on the chromosome are shown in Table 1:

[0007] Table 1 Positions of SNP molecular markers on the chromosome

[0008] 。

[0009] The present invention uses high-throughput sequencing to identify 33 SNP loci from the whole genome of pecan that can be used to identify or assist in identifying pecan varieties. These SNP loci can form a fingerprint map for the identification of 112 pecan varieties.

[0010] Furthermore, the sequences of the SNP loci in the above fingerprint map are shown as SEQ ID NO. 100 to SEQ ID NO. 132, and the SNP is located at the 201st position of any sequence of SEQ ID NO. 100 to SEQ ID NO. 132.

[0011] On the other hand, a fingerprint map for identifying pecan varieties, characterized in that the fingerprint map is composed of the aforementioned SNP molecular markers, the sequences of the SNP molecular markers are shown as SEQ ID NO. 100 to SEQ ID NO. 132, and the SNP is located at the 201st position of SEQ ID NO. 100 to SEQ ID NO. 132.

[0012] Those skilled in the art can understand that the fingerprint map 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. Therefore, the genotypes of 33 SNP loci of different varieties of the present invention can form different genotype barcodes, and the fingerprint map can also be considered to be composed of genotype barcodes of different pecan varieties.

[0013] On the other hand, the present invention provides KASP primers for detecting the aforementioned SNP molecular markers, and the primer sequences are shown as SEQ ID NO. 1 to SEQ ID NO. 99.

[0014] On the other hand, the present invention provides a kit for identifying or assisting in identifying pecan varieties, and the kit contains primers for detecting the aforementioned SNP molecular markers and / or fingerprint map.

[0015] On the other hand, another object of the present invention is to provide any one of the following applications of the aforementioned SNP molecular markers, fingerprint map, primers, and kit:

[0016] (1) Application in identifying pecan varieties;

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

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

[0019] Application in constructing the DNA fingerprint database of Carya illinoinensis

[0020] On the other hand, another object of the present invention is to provide a method for constructing a fingerprint map of Carya illinoinensis, characterized in that the method comprises the following steps:

[0021] (1) Extract the total DNA of Carya illinoinensis samples of different varieties;

[0022] (2) Detect the genotypes of SNP molecular markers in the samples. The sequences of the SNP molecular markers are shown in SEQ ID NO. 100 to SEQ ID NO. 132, and the mutation sites of the SNPs are located at the 201st position of the sequences of SEQ ID NO. 100 to SEQ ID NO. 132;

[0023] (3) Construct a fingerprint map of Carya illinoinensis according to the detection results.

[0024] Optionally, in step (2), the genotypes of the SNPs can be detected by sequencing, KASP or any other available genotyping technology.

[0025] On the other hand, another object of the present invention is to provide a method for identifying or assisting in identifying Carya illinoinensis varieties, characterized in that it includes:

[0026] (1) Extract the total DNA of the Carya illinoinensis sample to be identified;

[0027] (2) Detect the genotypes of each SNP molecular marker in the fingerprint map in the sample;

[0028] (3) Judge the variety of the Carya illinoinensis sample to be identified according to the detection results.

[0029] Optionally, in step (2), the genotypes of the aforementioned SNP molecular markers can be detected by sequencing, KASP or any other available genotyping technology.

[0030] The specific judgment method in step (3) is that when the genotype of the Carya illinoinensis sample to be identified is consistent with the genotype of any variety in the aforementioned fingerprint map, it is judged as the corresponding Carya illinoinensis variety.

[0031] On the other hand, the present invention provides any of the following applications of the aforementioned method:

[0032] (1) Application in identifying Carya illinoinensis varieties;

[0033] (2) Application in the identification, improvement or molecular marker-assisted breeding of Carya illinoinensis germplasm resources;

[0034] (3) Application in screening or creating different thin-shell pecan varieties;

[0035] (4) Application in constructing a DNA fingerprint database of thin-shelled pecan.

[0036] 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

[0037] 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.

[0038] Figure 1 It is a fingerprint map drawn from the KASP test results of 114 samples to be tested. DETAILED DESCRIPTION

[0039] 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.

[0040] 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.

[0041] Example 1 Resequencing Analysis

[0042] 1. Experimental Materials

[0043] The source of materials and information are shown in Table 2:

[0044] Table 2 Sources and information

[0045]

[0046] 2. Sample DNA extraction and library construction and sequencing

[0047] 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.

[0048] Specific experimental steps:

[0049] Construct the library with an initial DNA amount of 1 μg;

[0050] Extract high-quality genomic DNA using the CTAB method;

[0051] Detect the DNA fragment size and DNA degradation degree by 0.75% agarose gel electrophoresis;

[0052] Detect the DNA purity using a NanoDrop One spectrophotometer (Thermo Fisher Scientific), and the OD260 / 280 ratio is between 1.8 - 2.2, without protein and visible impurity contamination;

[0053] Detect the DNA concentration using a Qubit 3.0 fluorometer (Life Technologies, Carlsbad, CA, USA), and the detected concentration is greater than 50 ng / μl, with a total amount greater than 2 μg.

[0054] After Covaris M220 ultrasonic fragmentation of DNA, use magnetic beads for fragment selection to make the sample bands concentrated between 200 - 400 bp;

[0055] Arrange the qualified library for sequencing on the machine to perform 2 * 150 bp sequencing.

[0056] 3. Data quality control

[0057] (1) Remove the adapter sequences in the sequences, that is, the adapter;

[0058] (2) Remove polyG and polyX at the tail of the reads (the shortest length is 10 bp);

[0059] (3) Statistically calculate the average quality value of the bases within the window in a sliding window manner, and trim off the low-quality sliding windows, whose function is similar to Trimmomatic;

[0060] (4) Remove the reads with more than 5 Ns;

[0061] (5) Remove the reads with a proportion of bases with a quality lower than 15 higher than 40%;

[0062] (6) Remove the reads with a length lower than 15 bp after filtering.

[0063] 4. Data alignment

[0064] In the present invention, we use the genome of Carya illinoinensis as the reference genome, use the 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.

[0065] For each sample, bwa (version: 0.7.17; parameters: mem) alignment analysis was performed separately, aligning the filtered Clean reads to the reference genome and counting the alignment situation. The specific analysis steps are as follows:

[0066] (1) Using the bwa alignment software (parameters: mem -R, and the remaining parameters using the software default parameters), align the Clean reads of all samples to the reference genome;

[0067] (2) Use samtools (parameters: sort) to convert the alignment result from a sam (Sequence Alignment / MAP) file to a sorted bam file (binary Alignment / Map);

[0068] (3) Use samtools (parameters: markdup -r) to remove duplicates from the sorted alignment result for subsequent analysis;

[0069] (4) Use a python script to count the alignment rate and coverage.

[0070] 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 the filtered reads of all samples after aligning to the reference genome was 85.40% (90.44 - 97.44%).

[0071] A total of 31,365,448 SNP sites were developed.

[0072] Example 2 Development of core SNPs for identifying 112 pecan varieties

[0073] 1. Identification of core SNPs

[0074] According to the grouping among 112 pecan samples, select SNP combinations that can distinguish 112 pecan varieties. At the same time, to reduce the false positives of SNPs, the following analysis criteria are adopted:

[0075] 1. Site detection rate = 100%; 2. MAF > 0.15; 3. Heterozygosity rate < 0.4; 4. Remove redundant markers;

[0076] 5. There are no other SNPs within 30 bp before and after the selected SNP locus; 6. GC content: Analyze the GC content of the SNP locus to avoid selecting loci with too high or too low GC content. The GC content within 150 bp before and after the selected SNP locus is 40%-60%; 7. PIC >= 0.2; 8. Locus polymorphism >= 0.4; 9. There cannot be >= 8 consecutive single-base repeats within 30 bp upstream and downstream of the SNP locus; 10. There cannot be homology between 50 bp upstream and downstream of the SNP locus and other positions in the genome.

[0077] Finally, 33 SNPs were screened out. The genotypes of the 33 core SNP loci in different varieties are 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 combinations of these different SNPs constitute the fingerprint map.

[0078] Table 3 Multiple sequence alignment results of the bases of SNP loci of 112 samples arranged in ascending order according to SNP number

[0079]

[0080] Example 3 Molecular marker verification

[0081] 1) Extract the total DNA of the sample to be detected (the variety numbers of the independent verification samples are consistent with those in Example 1, and samples of non-Carya illinoinensis species numbered 113-114 are added);

[0082] 2) Primer and probe design;

[0083] Based on the SNP sites determined in Example 1, KASP primers and probes were designed based on the chromosomal sequence. The primer and probe sequences are shown in Table 4.

[0084] Table 4 KASP primers

[0085]

[0086] 3) Use the primers and probes designed in step 2) to perform KASP genotyping on the qualified DNA extracted in step 1). The primers, probes and combinations are prepared into a detection kit.

[0087] 4) Judge the variety of the sample according to the detection result.

[0088] The results are shown in Figure 1 , Figure 1 The fingerprint maps of show that the genotypes of 112 varieties are completely different, which is consistent with the resequencing results and is also completely different from the genotypes of samples No. 113 - 114.

[0089] In summary, the fingerprint maps prepared by the present invention can be used for the accurate identification of 112 varieties.

[0090] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to the above embodiments will be apparent to those skilled in the art. The general principles defined herein can 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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