A SNP molecular marker combination for identifying superior Liriodendron chinense strains and its application

Abstract

The present invention discloses a SNP molecular marker combination for identifying superior Liriodendron chinense strains and its application, belonging to the field of biological breeding technology. The SNP molecular marker for identifying superior Liriodendron chinense strains disclosed in the present invention consists of 10 SNP molecular markers, and each SNP molecular marker corresponds to a SNP site. Based on the results of Hyper‑seq high-throughput sequencing, the present invention has developed 10 pairs of SNP primers. Multiple primer combinations can be used to identify candidate superior strains and can be used to construct fingerprint maps of superior Liriodendron chinense strains. Superior Liriodendron chinense strains can be distinguished at the molecular level. This method has the advantages of high accuracy, good stability, simple operation, etc., and can play a positive role in Liriodendron chinense molecular marker-assisted breeding and variety protection.

Description

Technical Field

[0001] The present invention relates to a SNP molecular marker combination for identifying superior Liriodendron tulipifera strains and application thereof, belonging to the technical field of biological breeding. Background Art

[0002] Liriodendron chinense (Hemsl.) Sarg., also known as Liriodendron chinense, is a member of the genus Liriodendron in the Magnoliaceae family and a precious broad-leaved tree species unique to my country. Species of the genus Liriodendron include Liriodendron chinense and Liriodendron chinense, which are fast-growing deciduous trees with straight trunks, fine wood, beautiful flowers and leaves, few pests and diseases, and strong resistance to pollution. They have high timber, ornamental, and ecological value. Liriodendron chinense has a straight trunk and peculiar leaf shape. It has excellent characteristics such as fast growth and strong resistance. It is a precious broad-leaved tree species with excellent timber and ornamental properties, as well as carbon sequestration functions. The development and utilization of Liriodendron chinense resources can better tap into the resources of native broad-leaved tree species. The selection and application of fast-growing varieties of Liriodendron chinense will help improve the carbon sequestration capacity of forests, increase timber supply, and accelerate the improvement of forest quality.

[0003] Liriodendron tulipifera is an ancient relict tree species with a long evolutionary history and rich genetic variation, resulting in high levels of genetic diversity and heterogeneity. Research has been conducted both domestically and internationally on the collection and evaluation of Liriodendron genus germplasm resources and afforestation techniques. These studies have shown that growth traits vary significantly between provenances. Liriodendron tulipifera grows rapidly south of the Yangtze River basin, particularly in Jiangxi, Hunan, and Yunnan, and its superior germplasm can rival or even surpass hybrids. However, compared with hybrid and North American tulipifera, the development and utilization of superior Liriodendron tulipifera germplasm is relatively limited, with no high-quality varieties available. To fully tap the superior germplasm resources of Liriodendron tulipifera and cultivate high-quality varieties, 26 high-quality trees with fast growth and good trunk shape were initially selected based on their apparent growth morphology, using germplasm from 423 families of 53 provenances collected throughout the natural distribution of Liriodendron tulipifera. These 26 high-quality trees were then propagated to establish a clonal experimental forest. Through experimental observation, five excellent clones were initially selected: Goose Excellent 1, Goose Excellent 2, Goose Excellent 3, Goose Excellent 4, and Goose Excellent 5. These five excellent Liriodendron tulipifera plants (clones) have fast growth rates and straight trunks, and all have great potential application value.

[0004] At present, the identification of superior clones of Liriodendron chinense is mostly based on the appearance of bark color, leaf shape and color, and trunk shape. Although some clones can be distinguished by traditional means such as bark color, leaf shape and color, and trunk shape. However, many varieties and clones of Liriodendron chinense have similar appearance characteristics, and they need to be confirmed by multiple phenotypes such as growth rate, leaves, flowers and fruits after a certain number of years of growth. It is difficult to complete variety identification in the seedling stage, and there are certain problems with identification accuracy. Therefore, the development of an efficient and accurate technical means for the identification of superior Liriodendron chinense plants is of great significance to the protection and identification of superior clones of Liriodendron chinense during their promotion and application.

[0005] Molecular markers have evolved from the initial random amplified polymorphisms (RAPDs) to simple sequence repeats (SSRs), and now single nucleotide polymorphisms (SNPs) and gene chips. In recent years, SNPs have become a popular genetic marker, representing the third generation of molecular markers. SNPs primarily refer to DNA sequence polymorphisms caused by variations in a single nucleotide at the genomic level, and are widely present in plant genomes in the form of single base transversions, transitions, insertions, and deletions. SNP molecular markers have the characteristics of high genomic distribution density, easy data integration, and high detection throughput. Summary of the Invention

[0006] In view of the deficiencies in the prior art, the technical problem to be solved by the present invention is to provide a combination of SNP molecular markers for identifying superior strains of Liriodendron chinense. Another technical problem to be solved by the present invention is to provide a method for constructing a combination of SNP molecular markers for identifying superior strains of Liriodendron chinense. Another technical problem to be solved by the present invention is to provide a combination of PCR amplification primer pairs for the combination of SNP molecular markers for identifying superior strains of Liriodendron chinense. Another technical problem to be solved by the present invention is to provide an application of the combination of SNP molecular markers for identifying superior strains of Liriodendron chinense, for quickly and accurately identifying superior strains of Liriodendron chinense and constructing a fingerprint for identifying superior strains of Liriodendron chinense.

[0007] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0008] A SNP molecular marker combination for identifying superior Liriodendron chinense strains, the SNP molecular marker combination consisting of 10 SNP molecular markers, each SNP molecular marker corresponding to a SNP site:

[0009] The first SNP molecular marker corresponds to SNP site SNP1:3_3942875;

[0010] The second SNP molecular marker corresponds to the SNP site SNP2: 13_5289469;

[0011] The third SNP molecular marker corresponds to the SNP site SNP3: 32_4487946;

[0012] The fourth SNP molecular marker corresponds to SNP site SNP4: 47_1965761;

[0013] The fifth SNP molecular marker corresponds to the SNP site SNP5: 130_10969799;

[0014] The sixth SNP molecular marker corresponds to the SNP site SNP6: 172_4190630;

[0015] The seventh SNP molecular marker corresponds to the SNP site SNP7: 288_797964;

[0016] The eighth SNP molecular marker corresponds to the SNP site SNP8: 1380_1213726;

[0017] The ninth SNP molecular marker corresponds to the SNP site SNP9: 1400_2342608;

[0018] The tenth SNP molecular marker corresponds to SNP site SNP10: 1402_342747.

[0019] The method for constructing the SNP molecular marker combination specifically comprises the following steps:

[0020] 1) Extracting total DNA from Liriodendron chinense;

[0021] 2) The extracted total DNA of Liriodendron chinense was subjected to Hyper-seq high-throughput sequencing and the data was aligned to the Liriodendron chinense genome;

[0022] 3) SNP and INDEL data of each sample were statistically analyzed to obtain variant identification results. Highly diverse variant sites were selected to construct fingerprint maps of each sample, and finally a SNP molecular marker combination for identifying superior Liriodendron chinense strains was obtained.

[0023] Application of a SNP molecular marker combination for identifying superior Liriodendron chinense strains in identifying superior Liriodendron chinense strains and / or constructing a fingerprint for identifying superior Liriodendron chinense strains and / or preparing a kit for identifying superior Liriodendron chinense strains.

[0024] The PCR amplification primer pair combination of the SNP molecular marker combination for identifying superior Liriodendron chinense strains includes:

[0025] SNP1-F: 5'-GCGGGCTACTCCCTATTCAC-3',

[0026] SNP1-R: 5'-CCGCCACCATCGAAATTCAC-3';

[0027] SNP2-F:5’-TGGGCTCAACGTCTTCACAA-3’,

[0028] SNP2-R:5’-GGGAATACCGGAACGAAGCA-3’;

[0029] SNP3-F:5’-CCCTGTTCACGGTCTGTTGT-3’,

[0030] SNP3-R:5’-CAATCATGCGCAGCTTGTGA-3’;

[0031] SNP4-F:5’-ACGTGTGAGATGCTAGTGGC-3’,

[0032] SNP4-R:5’-ACTTCCATCTCTCCCCAGCA-3’;

[0033] SNP5-F:5’-CCAGCTCAAGCCTAAACCCA-3’,

[0034] SNP5-R:5’-AAAGGGATGGAGCTGGTTCG-3’;

[0035] SNP6-F:5’-GTCTAGGAACTCGCTGCCAT-3’,

[0036] SNP6-R:5’-ACGATGTCAACAATGTACAACCC-3’;

[0037] SNP7-F:5’-GAAGTCCTGCTGCTAGTGGG-3’,

[0038] SNP7-R:5’-CCCCACCCCTTTCATCTCAC-3’;

[0039] SNP8-F:5’-GTGAGAGGGAGCTTGGGTTT-3’,

[0040] SNP8-R:5’-TTGGCACGTTCGGACACTAA-3’;

[0041] SNP9-F:5’-ATCTCCTCTCATGTGGCCCT-3’,

[0042] SNP9-R:5’-CTGCGACTGAAATGATGGCG-3’;

[0043] SNP10-F: 5'-GGCTTGCACTTTCTCAGCAC-3',

[0044] SNP10-R: 5'-ACCTGAAACCAGCAAACCCT-3'.

[0045] The PCR amplification primer pair combination is used in identifying superior Liriodendron chinense strains.

[0046] The application of the PCR amplification primer pair combination in constructing a fingerprint map for identifying superior Liriodendron chinense strains.

[0047] The PCR amplification primer pair combination is used in preparing a kit for identifying superior Liriodendron chinense strains.

[0048] Beneficial effects of the present invention:

[0049] The SNP molecular markers disclosed in the present invention for identifying superior Liriodendron tulipifera strains consist of 10 SNP molecular markers, each corresponding to a single SNP locus. Based on the results of Hyper-seq high-throughput sequencing, the present invention developed 10 pairs of SNP primers. Multiple primer combinations can be used to identify candidate superior strains and construct fingerprints of superior Liriodendron tulipifera strains, enabling differentiation of superior Liriodendron tulipifera strains at the molecular level. This method has the advantages of high accuracy, good stability, and simple operation, and can play a positive role in molecular marker-assisted breeding and variety protection of Liriodendron tulipifera. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 The fingerprint maps of 11 individuals and superior strains of Liriodendron chinense. DETAILED DESCRIPTION

[0051] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0052] The five Liriodendron tulipifera, one Liriodendron tulipifera, and five superior Liriodendron tulipifera individuals used in this application were all planted in the Lishan Forest Farm in Yongfeng County. The five Liriodendron tulipifera and one Liriodendron tulipifera were randomly selected. Two to three fresh young leaves were collected from each sample, dried in a sealed bag containing silica gel, and stored in a refrigerator at -80°C. See Table 1.

[0053] Table 1 Eleven individuals and superior strains of Liriodendron chinense used in this application

[0054]

[0055]

[0056] Example 1

[0057] 1. DNA extraction

[0058] The total DNA of Liriodendron chinense leaves was extracted using a plant genomic DNA extraction kit (Kangwei Reagent Biotechnology Co., Ltd.).

[0059] 2. Development of polymorphic sites

[0060] The extracted DNA from all samples was homogenized to a concentration of 150-200 ng / μL. Hyper-seq libraries were constructed for all 11 samples. After passing the assay, high-throughput sequencing was performed using the second-generation Illumina NovaSeq 6000 platform or the DNBSEQ-T7 platform (Illumina, San Diego, CA, USA). After sequence quality control, the data were aligned to the genome of the red tulip tree.

[0061] The results showed that the known reference genome size of Liriodendron chinense is 1.74G, the alignment rate of 11 samples to the Liriodendron chinense genome was between 99.08% and 99.82%, the coverage of the reference genome was between 2.856% and 4.515%, and the average sequencing depth was between 0.413X and 0.88X.

[0062] 3. Construction of fingerprint map of Liriodendron chinense

[0063] Based on the variant identification results, the diallelic SNP sites with a maf value (minor allele frequency) greater than 0.05 and a genotype missing rate of 0 were retained, and finally 10 variant sites with high diversity were selected (Table 2).

[0064] Table 2 Information of 10 SNP sites

[0065] Site chromosome Location Basetype 1 Base type 2 SNP1 Scaffold3 3942875 G A SNP2 Scaffold13 5289469 A T SNP3 Scaffold32 4487946 G A SNP4 Scaffold47 1965761 T C SNP5 Scaffold130 10969799 C T SNP6 Scaffold172 4190630 G A SNP7 Scaffold288 797964 A G SNP8 Scaffold1380 1213726 C T SNP9 Scaffold1400 2342608 G A SNP10 Scaffold1402 342747 T G

[0066] The base information of these 10 SNP polymorphic sites was used to construct fingerprint maps of 5 superior strains, 5 Liriodendron chinense and 1 Liriodendron chinense (Table 3).

[0067] Table 3 Fingerprints of 11 superior strains of Liriodendron chinense

[0068] Name of superior strain Fingerprint Liriodendron 1 0102000000 Liriodendron 2 0100002010 Goose 1 0000000001 Goose 2 0012000000 Liriodendron 3 0022000010 Goose 3 0002102010 Goose 4 2000100020 Liriodendron tulipifera 1012000000 Liriodendron 4 0000001220 Goose 5 0102020000 Liriodendron 5 0001001022

[0069] Among them, 0 means that all SNP sites are base type 1, 1 means that two base types coexist, and 2 means that all SNP sites are base type 2.

[0070] Different colors are assigned to different base types at the 10 SNP sites of each sample to visualize the fingerprint map.

[0071] The results are as follows Figure 1 As shown, the five Liriodendron chinense varieties have high genetic similarity, but there are still suitable loci for differentiation.

[0072] 4. Finally, it was determined that the combination of 10 SNP molecular markers could distinguish different superior strains of Liriodendron tulipifera and was used to construct a DNA fingerprint of Liriodendron tulipifera. The nucleotide sequences of the 10 SNP molecular markers corresponded to the nucleotide sequences of SEQ ID NOs. 1-10, respectively.

[0073] Example 2

[0074] 1. Design and synthesis of SNP site primers

[0075] Primers were designed using the Primer-BLAST software obtained from the NCBI website. Each pair of primers was required to be located at least 50 bp from the polymorphic site, and the product length was set between 200 and 600 bp. A total of 10 pairs of SNP primers were designed (Table 4) and synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0076] Table 4 10 pairs of SNP site amplification primers

[0077]

[0078]

[0079] 2. Screening of SNP primers for identification of superior Liriodendron chinense strains and product detection

[0080] The genomic DNAs of the above-mentioned five common tulip trees, five superior strains and one Liriodendron tulipifera were used as templates, and amplification was performed using the 10 pairs of SNP primers in Table 4 to obtain PCR amplification products.

[0081] PCR reaction system (20 μL): 10 μL of 2× Bio Taq PCR Mix (+Dye) Mix, 40 ng of DNA template, 0.2 μM of each upstream and downstream primers, and ddH2O to make up to 20 μL.

[0082] TouchDown PCR reaction program: pre-denaturation at 95°C for 5 min; 15 cycles of denaturation at 95°C for 30 s, annealing at 65-55°C (0.7°C decrease per cycle) for 30 s, and extension at 72°C for 40 s; 20 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 40 s; and extension at 72°C for 4 min.

[0083] PCR products were examined on a 1.2% agarose gel at 120 V for 15-20 min. Clear bands were visible for primer amplification, and the fragment size was consistent with the target fragment size.

[0084] The amplified PCR product containing the target fragment was sent to Shanghai Sangon Biotechnology Co., Ltd. and sequenced using the amplified primer product.

[0085] Sequencing revealed that the sequencing results of the 10 SNP sites were consistent with the initial Hyperseq sequencing results. The length of the SNP site amplification product and the base of the polymorphic site are shown in Table 5.

[0086] Table 5 Information of 10 SNP sites

[0087]

[0088]

[0089] Example 3

[0090] Five samples of Liriodendron chinense, five superior strains, and one sample of Liriodendron tulipifera were randomly selected and numbered 1-11. Genomic DNA was extracted from each sample and sequence amplification was performed using 10 pairs of primers targeting SNP loci (Table 4).

[0091] PCR reaction system (20 μL): 10 μL of 2× Bio Taq PCR Mix (+Dye) Mix, 40 ng of DNA template, 0.2 μM of each upstream and downstream primers, and ddH2O to make up to 20 μL.

[0092] TouchDown PCR reaction protocol: 95°C initial denaturation for 5 min; 15 cycles of 95°C denaturation for 30 s, 65-55°C (0.7°C decrease per cycle) annealing for 30 s, and 72°C extension for 40 s; 20 cycles of 95°C denaturation for 30 s, 55°C annealing for 30 s, and 72°C extension for 40 s; and 72°C extension for 4 min. PCR amplification products were sent to Shanghai Sangon Biotechnology Co., Ltd. for next-generation sequencing using the amplified primer sequences.

[0093] Based on the base sequencing results of different samples at the 10 SNP sites, they were converted into fingerprint codes for identification and verification. The specific identification results are shown in Table 6.

[0094] Table 6 Identification results of 11 Liriodendron chinense samples

[0095] Sample Code Fingerprint Identification results Actual samples Sample 1 2000100020 Goose 4 Goose 4 Sample 2 0001001022 Liriodendron 5 Liriodendron 5 Sample 3 0012000000 Goose 2 Goose 2 Sample 4 0100002010 Liriodendron 2 Liriodendron 2 Sample 5 0000000001 Goose 1 Goose 1 Sample 6 0002102010 Goose 3 Goose 3 Sample 7 0022000010 Liriodendron 3 Liriodendron 3 Sample 8 1012000000 Liriodendron tulipifera Liriodendron tulipifera Sample 9 0000001220 Liriodendron 4 Liriodendron 4 Sample 10 0102000000 Liriodendron 1 Liriodendron 1 Sample 11 0102020000 Goose 5 Goose 5

[0096] Example 4

[0097] A kit for identifying superior strains of Liriodendron chinense, comprising the following molecular marker combination:

[0098] The first SNP molecular marker, whose nucleotide sequence is shown in SEQ ID NO.1, wherein R is G or A; the second SNP molecular marker, whose nucleotide sequence is shown in SEQ ID NO.2, wherein W is A or T; the third SNP molecular marker, whose nucleotide sequence is shown in SEQ ID NO.3, wherein R is G or A; the fourth SNP molecular marker, whose nucleotide sequence is shown in SEQ ID NO.4, wherein Y is T or C; the fifth SNP molecular marker, whose nucleotide sequence is shown in SEQ ID NO.5, wherein Y is C or T; the sixth SNP molecular marker, whose nucleotide sequence is shown in SEQ ID NO.6, wherein R is G or A; the seventh SNP molecular marker, whose nucleotide sequence is shown in SEQ ID NO.7, wherein R is A or G; the eighth SNP molecular marker, whose nucleotide sequence is shown in SEQ ID NO.8, wherein Y is C or T; the ninth SNP molecular marker, whose nucleotide sequence is shown in SEQ ID NO.9, wherein R is G or A; the tenth SNP molecular marker, whose nucleotide sequence is shown in SEQ ID NO.10 As shown in NO.10, wherein K is T or G.

[0099] Each SNP molecular marker corresponds to a SNP site. The first SNP molecular marker corresponds to SNP1 (3_3942875), the second SNP molecular marker corresponds to SNP2 (13_5289469), the third SNP molecular marker corresponds to SNP3 (32_4487946), the fourth SNP molecular marker corresponds to SNP4 (47_1965761), the fifth SNP molecular marker corresponds to SNP5 (130_10969799), the sixth SNP molecular marker corresponds to SNP6 (172_4190630), the seventh SNP molecular marker corresponds to SNP7 (288_797964), the eighth SNP molecular marker corresponds to SNP8 (1380_1213726), the ninth SNP molecular marker corresponds to SNP9 (1400_2342608), and the tenth SNP molecular marker corresponds to SNP10 (1402_342747).

[0100] Each SNP site corresponds to the corresponding SNP primer shown in Table 4.

[0101] This makes it possible to simply and accurately identify different superior Liriodendron chinense strains under different environments, different periods and different growing seasons.

Claims

1. Application of a PCR amplification primer pair combination of a SNP molecular marker combination for identifying superior Liriodendron chinense strains in identifying superior Liriodendron chinense strains; the PCR amplification primer pair combination is: SNP1-F: 5'-GCGGGCTACTCCCTATTCAC-3', SNP1-R: 5'-CCGCCACCATCGAAATTCAC-3'; SNP2-F: 5'-TGGGCTCAACGTCTTCACAA-3', SNP2-R: 5'-GGGAATACCGGAACGAAGCA-3'; SNP3-F: 5'-CCCTGTTTCACGGTCTGTTGT-3', SNP3-R: 5'-CAATCATGCCGCAGCTTGTGA-3'; SNP4-F: 5'-ACGTGTGAGATGCTAGTGGC-3', SNP4-R: 5'-ACTTCCATCTCTCCCCAGCA-3'; SNP5-F: 5'-CCAGCTCAAGCCTAAACCCA-3', SNP5-R: 5'-AAAGGGATGGAGCTGGTTCG-3'; SNP6-F: 5'-GTCTAGGAACTCGCTGCCAT-3', SNP6-R: 5'-ACGATGTCAACAATGTACAACCC-3'; SNP7-F: 5'-GAAGTCCTGCTGCTAGTGGG-3', SNP7-R: 5'-CCCCACCCCTTTCATCTCAC-3'; SNP8-F: 5'-GTGAGAGGGAGCTTGGGTTT-3', SNP8-R: 5'-TTGGCACGTTCGGACACTAA-3'; SNP9-F: 5'-ATTCTCCTCTCATGTGGCCCT-3', SNP9-R: 5'-CTGCGACTGAAATGATGGCG-3'; SNP10-F: 5'-GGCTTGCACTTTCTCAGCAC-3', SNP10-R: 5'-ACCTGAAACCAGCAAACCCT-3'; The application process is as follows: 1) extracting DNA from a sample to be tested, and performing PCR amplification using the combination of PCR amplification primers; 2) Sequencing the amplified products to obtain base sequencing results at 10 SNP sites for different samples; The base sequencing results at the 10 SNP sites are: ； 3) Based on the base sequencing results of different samples at 10 SNP sites, they are converted into fingerprint codes to complete the identification; The standard sample fingerprint code is as follows: The superior strain name is Liriodendron chinense 1, and its fingerprint is: 0102000000; The superior strain name is Liriodendron chinense 2, and the fingerprint is: 0100002010; The name of the superior strain is: Goose Excellent 1, and the fingerprint is: 0000000001; The name of the superior strain is: Goose Excellent 2, and the fingerprint is: 0012000000; The superior strain name is Liriodendron chinense 3, and the fingerprint is: 0022000010; The name of the superior strain is: Goose Excellent 3, and the fingerprint is: 0002102010; The name of the superior strain is: Goose Excellent 4, and the fingerprint is: 2000100020; The superior strain name is Liriodendron tulipifera, and the fingerprint is: 1012000000; The superior strain name is Liriodendron 4, and the fingerprint is: 0000001220; The name of the superior strain is: Goose Excellent 5, and the fingerprint is: 0102020000; The superior strain name is Liriodendron 5, and the fingerprint is: 0001001022; in, 0 means that all SNP sites are base type 1, 1 means that both base types coexist, and 2 means that all SNP sites are base type 2.

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