SNP molecular marker for identifying carya illinoinensis cultivar'mahal' and application thereof

Abstract

The present application relates to the technical field of molecular marker, in particular to a SNP molecular marker of 'Maham' Pecan and application thereof. The SNP molecular marker can be used to identify 'Maham' Pecan, and has important significance for germplasm resource identification and molecular marker breeding of Pecan.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a SNP molecular marker for identifying Carya illinoinensis variety 'Maham' and application thereof. BACKGROUND

[0002] Carya illinoinensis, a deciduous large tree of Carya in Juglandaceae, is also called American Carya and originates from the United States. Carya illinoinensis is a world-famous ecological and economic tree species due to its delicious and non-astringent fruits, high yield, straight trunk and excellent wood quality. The kernel of Carya illinoinensis is rich in unsaturated fatty acids such as oleic acid and linoleic acid, as well as proteins, polyphenols, flavonoids and trace elements, and has the effects of benefiting the brain and intelligence, delaying aging and preventing cardiovascular and cerebrovascular diseases, and is therefore also called longevity fruit and is deeply loved by the public.

[0003] Carya illinoinensis was introduced and cultivated in China in 1900. With the breakthrough in breeding technology in recent years, the planting area is continuously expanding. However, with the continuous heating of the seedling consumption market, there are too many cases of "same thing with different names" and "same name with different things". There are also cases of using inferior seedlings to replace good ones or even using seedlings of other species to fill in the blanks.

[0004] 'Maham' is introduced from an excellent variety in the United States. The nut is narrow and long, with a longitudinal diameter of about 4 cm, an oblong shape, an asymmetric acute angle at the top of the fruit, a round base, and a concave middle part. The fruit matures in mid-October. The shell is medium in thickness, with an average thickness of about 0.75 mm, and is easy to peel. The kernel is golden yellow. Although 'Maham' has many advantages and differences compared with other Carya illinoinensis varieties, it is still difficult for ordinary technical personnel to efficiently identify the variety during the non-fruiting period, especially during the seedling stage. Moreover, only phenotypic data is relatively subjective, and it is more convincing to combine molecular biology (such as fingerprinting technology) evidence. DNA barcodes developed based on molecular marker technology have high throughput and sensitivity, and can accurately identify 'Maham' on a large scale. SUMMARY

[0005] The present application mainly aims at the above technical problems, and provides a simple molecular marker method to identify 'Maham' Carya illinoinensis. Specifically, the present application provides the following technical solutions:

[0006] In a first aspect, the present application provides a DNA barcode for identifying or assisting in identifying 'Maham' Carya illinoinensis variety, characterized in that the sequence of the DNA barcode is GRCGSTR, and the base positions of the DNA barcode on the chromosome are shown in the following table in order:

[0007] .

[0008] The present application identifies 7 SNP loci from the whole genome of Carya illinoensis which can be used to identify or assist in identifying the ‘Mahahn’ variety of Carya illinoensis, which can form a DNA barcode unique to the ‘Mahahn’ variety. Those skilled in the art can understand that the DNA barcode of the present application is not formed by consecutive bases, but by the genotypes of SNPs scattered in different loci of different chromosomes, so the DNA barcode of the present application can also be referred to as a genotype barcode.

[0009] Further, the sequences of the SNP loci in the above DNA barcode are shown in SEQ ID NO. 22~SEQ ID NO. 28 (in order of the positions of the SNPs in the DNA barcode):

[0010] .

[0011] In particular, the genotype of locus CHR1_6071055 (chromosome 1, locus 6071055) is GG, which is different from all other varieties. Since the propagation of the varieties of Carya illinoensis after breeding is single plant asexual propagation to ensure the stability of the variety traits, the genotype of locus CHR1_6071055 is GG, which can be used as a unique locus of ‘Mahahn’ and can be used for identification or assisted identification of the variety.

[0012] Further, the sequence of the SNP molecular marker CHR1_6071055 is shown in SEQ ID NO. 22, the SNP molecular marker CHR1_6071055 is an A / G mutation at position 201 of SEQ ID NO. 22, and the genotype of the SNP molecular marker CHR1_6071055 in the ‘Mahahn’ variety of Carya illinoensis is GG.

[0013] In another aspect, the present application provides KASP primers for detecting the aforementioned SNP molecular markers, and the sequences of the primers are shown in SEQ ID NO. 1~21.

[0014] .

[0015] In another aspect, the present application provides a kit for identifying or assisting in identifying the ‘Mahahn’ variety of Carya illinoensis, which comprises primers and / or probes for detecting the aforementioned DNA barcode and / or SNP molecular markers.

[0016] In another aspect, another object of the present application is to provide any of the following applications of the aforementioned DNA barcode, molecular marker, primer, and kit:

[0017] (1) for identifying the ‘Mahahn’ variety of Carya illinoensis;

[0018] (2) In the application of Carya illinoinensis germplasm identification, improvement or molecular marker assisted breeding;

[0019] (3) In the application of screening or creating different Carya illinoinensis varieties;

[0020] (4) In the application of constructing Carya illinoinensis DNA fingerprint database.

[0021] On the other hand, another object of the present application is to provide another method for identifying or assisting in identifying Carya illinoinensis variety ‘Mahahn’, characterized in that it comprises:

[0022] (1) extracting total DNA of the Carya illinoinensis sample to be identified;

[0023] (2) detecting the genotype of each base of the aforementioned DNA barcode;

[0024] (3) judging the variety of the Carya illinoinensis sample to be identified according to the detection result.

[0025] Optionally, step 2) can detect the genotype of each base of the aforementioned DNA barcode by sequencing or KASP or any other available genotyping technology.

[0026] The specific judging method of step 3) is that when the genotype of the Carya illinoinensis sample to be identified is inconsistent with the aforementioned DNA barcode, it is judged as not being the Carya illinoinensis variety ‘Mahahn’.

[0027] Further, for step 3), when the genotype of the Carya illinoinensis sample to be identified is consistent with the aforementioned DNA barcode, it is judged as the Carya illinoinensis variety ‘Mahahn’.

[0028] On the other hand, another object of the present application is to provide another method for identifying or assisting in identifying Carya illinoinensis variety ‘Mahahn’, characterized in that it comprises:

[0029] (1) extracting total DNA of the Carya illinoinensis sample to be identified;

[0030] (2) detecting the genotype of each base of the aforementioned DNA barcode;

[0031] (3) judging the variety of the Carya illinoinensis sample to be identified according to the detection result.

[0032] Optionally, step 2) can detect the genotype of each base of the aforementioned DNA barcode by sequencing or KASP or any other available genotyping technology.

[0033] The specific judging method of step 3) is that when the genotype of the Carya illinoensis sample to be identified and the genotype of the aforementioned SNP molecular marker CHR1_6071055 are inconsistent, it is judged as not being the Carya illinoensis variety 'Mahahn'.

[0034] Further, step 3) is that when the genotype of the Carya illinoensis sample to be identified and the genotype of the SNP molecular marker CHR1_6071055 are consistent, it is judged as the Carya illinoensis variety 'Mahahn'.

[0035] The aforementioned identification or auxiliary identification means that the sample to be detected is directly identified as the Carya illinoensis variety 'Mahahn', or only as an auxiliary means to exclude the sample to be identified as the Carya illinoensis variety 'Mahahn'.

[0036] Further, the aforementioned identification or auxiliary identification method can at least distinguish 'Mahahn' from any other variety described in the present application.

[0037] In another aspect, the present application provides any of the following applications of the aforementioned method:

[0038] (1) application in identifying the Carya illinoensis variety 'Mahahn';

[0039] (2) application in identifying, improving or molecular marker assisted breeding of Carya illinoensis germplasm resources;

[0040] (3) application in screening or creating different Carya illinoensis varieties;

[0041] (4) application in constructing a DNA fingerprint database of Carya illinoensis.

[0042] The present application has the following beneficial effects: a single DNA barcode or SNP molecular marker can effectively distinguish whether the variety to be detected is the Carya illinoensis variety 'Mahahn', providing a guarantee for identification, planting, resource utilization and breeding of the Carya illinoensis variety 'Mahahn'. It also has important significance for molecular marker breeding of Carya illinoensis. BRIEF DESCRIPTION OF DRAWINGS

[0043] The method of the present application and its beneficial effects will be described in detail below in combination with the drawings and specific embodiments.

[0044] Figure 1 is the detection result of 112 samples to be detected.

[0045] Figure 2 is the genotyping result of the CHR1_6071055 site of 114 samples, and the samples are arranged in order according to the number from the first row to the right (the last black square has no sample); wherein Figure A is sample No. 1~94, and Figure B is sample No. 95~114. DETAILED DESCRIPTION

[0046] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work belong to the scope of protection of the present application.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The Carya illinoinensis plant samples used in the following examples can be obtained by commercially available means or collected from the wild.

[0048] Resequencing analysis of Example 1

[0049] 1. Experimental materials

[0050] The sources and information of the materials are shown in Table 1:

[0051] Table 1. Sources and information of materials (notes are different names for the same sample)

[0052]

[0053] 2. Sample DNA extraction and library sequencing

[0054] First, the leaf samples were stored in liquid nitrogen, and the genomic DNA of samples 1-112 was extracted using a kit. A total of 994 Gb of raw data was obtained by sequencing, and the sequencing results were 150 bp paired-end data.

[0055] Specific experimental steps:

[0056] Library construction with 1 μg of DNA as the starting amount;

[0057] High-quality genomic DNA was extracted by the CTAB method;

[0058] 0.75% agarose gel electrophoresis was used to detect the size of the DNA fragments and the degree of DNA degradation;

[0059] NanoDrop One spectrophotometer (Thermo Fisher Scientific) was used to detect the DNA purity, and the OD260 / 280 ratio was between 1.8-2.2, without protein and visible impurity contamination;

[0060] Qubit 3.0 fluorescence instrument (Life Technologies, Carlsbad, CA, USA) was used to detect the DNA concentration, and the concentration was greater than 50 ng / μl, and the total amount was greater than 2 ug.

[0061] After the DNA is broken by Covaris M220 ultrasonic, the fragments are selected by magnetic beads so that the sample bands are concentrated between 200-400bp;

[0062] The qualified library is arranged for machine, and 2*150bp sequencing is performed.

[0063] 3. Data quality control

[0064] (1) The adapter sequence in the sequence is removed, that is, the adapter;

[0065] (2) The polyG and polyX (the minimum length is 10bp) at the tail of the read are removed;

[0066] (3) The average quality value of the bases in the window is calculated in a sliding window manner, and the low-quality sliding window is cut off, which is similar to Trimmomatic;

[0067] (4) The reads with N number greater than 5 are removed;

[0068] (5) The reads with the proportion of bases with quality less than 15 higher than 40% are removed;

[0069] (6) The reads with length less than 15bp after filtering are removed.

[0070] 4. Data alignment

[0071] In the application, the sequencing fragments are aligned back to the reference genome by using BWA alignment software, and then Picard-tools is used to remove the sequencing fragments generated by PCR-duplication.

[0072] Each sample is subjected to bwa (version: 0.7.17; parameter: mem) alignment analysis, and the filtered Clean reads are aligned to the reference genome to count the alignment conditions. The specific analysis steps are as follows:

[0073] (1) The Clean reads of all samples are aligned to the reference genome by using bwa alignment software (parameter: mem -R, and the remaining parameters are the default parameters of the software);

[0074] (2) The alignment results are converted from sam (Sequence Alignment / MAP) file to sorted bam (binary Alignment / Map) file by using samtools (parameter: sort);

[0075] (3) Remove duplicates from the sorted alignment using samtools (parameter: markdup -r) for subsequent analysis;

[0076] (4) Calculate the mapping rate and coverage.

[0077] The average sequencing depth of the samples was 10.42x (9.29-26.08x) based on the reference genome. After filtering, the average genome coverage of each sample was 85.40% (90.44-97.44%) after aligning to the reference genome.

[0078] A total of 31,365,448 SNP sites were developed.

[0079] Example 2 Development of core SNPs for identifying ‘Mahahn’

[0080] 1. Identification of core SNPs

[0081] Based on the grouping of ‘Mahahn’ and other P. monticola, SNPs that distinguish ‘Mahahn’ and other P. monticola were selected. The selection criteria were that no more than 50% of the sites had the same genotype as ‘Mahahn’, and that the combination could identify ‘Mahahn’. In order to reduce false positives of SNPs, the following analysis criteria were adopted:

[0082] 1. No other SNPs within 30bp upstream and downstream of the SNP site;

[0083] 2. No homology within 50bp upstream and downstream of the SNP site and other positions in the genome;

[0084] 3. No >=8 consecutive single base repeats within 30bp upstream and downstream of the SNP site;

[0085] 4. The GC content of 150bp before and after the selected SNP site is between 40%-60%;

[0086] 5. Extract 250bp sequences upstream and downstream of the remaining markers to design primers, product size: 250-400; Design primers that align to the genome, filter primers that can align to multiple places.

[0087] Finally, 7 markers were selected as core SNPs, and the 7 core SNP sites were used as the core SNP sites for identifying ‘Mahahn’. The genotypes of the 7 core SNP sites in different varieties were ranked from small to large according to the chromosome number and position, and the results are shown in Table 2. The specific ranking order was CHR1_6071055, CHR1_52228116, CHR5_10451065, CHR7_35325592, CHR8_8984149, CHR13_19946268, and CHR15_19954498.

[0088] The genotype combination of the 7 sites in ‘Mahahn’ was ranked into a barcode sequence as GRCGSTR, which was completely different from the SNP genotype combination of other varieties (Table 2), and could be used as a DNA barcode for identifying ‘Mahahn’. In particular, the genotype of site CHR1_6071055 was GG, which was different from all other varieties. Considering that the propagation of pecan varieties after breeding is single asexual propagation to ensure the stability of the variety traits. Therefore, the site CHR1_6071055 with GG genotype can be considered as a unique site of ‘Mahahn’, which can be used for the identification of the variety.

[0089]

[0090] Example 3 Verification of molecular markers

[0091] 1) Extract total DNA from the samples to be detected (independent verification sample variety number and consistent with Example 1, and add 113-114 non-Carya illinoinensis species samples);

[0092] 2) Design of primers and probes;

[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]

[0095] 3) Use the primers and probes designed in step 2) to detect the DNA extracted in step 1) using KASP for genetic detection typing. The primers and probes are combined to prepare a detection kit.

[0096] 4) Determine the variety of the sample according to the detection results.

[0097] The results are shown in Figures 1-2 , Figure 1 The results are shown in Figure 2The CHR1_6071055 site of the 'Mahar' variety is shown to be completely different from other samples.

[0098] In summary, the DNA barcode prepared by the present application can be used for accurate identification of the 'Mahar' variety, and the SNP site CHR1_6071055 alone can directly identify the 'Mahar' variety.

[0099] The above description of disclosed embodiments enables one of ordinary skill in the art to make or use the application. Various modifications to the above-described embodiments will be apparent to those of ordinary skill in the art, and the generic principles defined herein can be applied to other embodiments without departing from the spirit or scope of the application. Therefore, the present application is not intended 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 CHR1_6071055 for identifying the 'Mahan' thin-shelled pecan variety, characterized in that, The sequence of the SNP molecular marker CHR1_6071055 is shown in SEQ ID NO.

25. The SNP molecular marker CHR1_6071055 is an A / G mutation located at position 201 of SEQ ID NO.

25. The genotype of the SNP molecular marker CHR1_6071055 in the 'Mahan' thin-shelled pecan variety is GG.

2. A SNP molecular marker for identifying the 'Mahan' thin-shelled pecan variety, characterized in that... , The sequences of the SNP molecular markers are shown in SEQ ID NO.22~SEQ ID NO.28, 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 sequences of the KASP primers for detecting the SNP molecular marker of claim 1 are shown in SEQ ID NO.1~SEQ ID NO.3, and the sequences for detecting the SNP molecular marker of claim 2 are shown in SEQ ID NO.1~SEQ ID NO.

21.

4. A kit for identifying the 'Mahan' 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 molecular marker of claim 2, the primer of claim 3, or the kit of claim 4: (1) Application in identifying the 'Mahan' 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 'Mahan' 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 CHR1_6071055 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 SNP molecular marker CHR1_6071055 of claim 1 can be detected by sequencing, KASP or any other available genotyping technique.

8. A method for identifying the 'Mahan' 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 'Mahan' 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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