SNP (Single Nucleotide Polymorphism) molecular marker for rapidly identifying high-aroma perfume coconut

Abstract

The invention provides an SNP (Single Nucleotide Polymorphism) molecular marker for rapidly identifying high-aroma perfume coconuts, the sequence of the SNP marker is as shown in SEQ ID NO: 1, and the basic group at the 98bp site from the 5'end of the sequence as shown in SEQ ID NO: 1 is C or T. The invention provides an SNP (Single Nucleotide Polymorphism) molecular marker for quickly identifying high-aroma type perfume coconuts, and designs a group of SNP molecular marker primer combinations for specifically identifying the germplasm of the high-aroma type perfume coconuts, so that the molecular identification of the germplasm of the high-aroma type perfume coconuts and the germplasm of common type coconuts can be quickly and accurately realized. Compared with a traditional method, the technical scheme provided by the invention has the remarkable advantages of simplicity and convenience in operation, short detection period (identification can be completed in a seedling stage), objective and reliable result and the like, and provides an efficient technical means for germplasm resource evaluation, molecular marker-assisted breeding and commercial variety identification of perfume coconuts; the method has great potential application value in the coconut industry.

Description

Technical Field

[0001] This invention relates to an SNP marker and its application, and more particularly to an SNP molecular marker for rapid identification of highly aromatic perfume coconut. Background Technology

[0002] coconut( Cocos nucifera Coconut (L.) is one of the most important economic crops in tropical regions, with a cultivation history of over 2000 years in Hainan Province, China. Like many other agricultural products, the flavor of raw coconut is the most important factor for farmers, plantations, and processing enterprises in raw coconut production. Flavor, as a crucial sensory quality of coconut, has always been a goal pursued by breeders during long-term domestication and improvement. The fragrant coconut is a specific mutant type of dwarf coconut, with a unique aroma in its coconut juice similar to taro, making it very popular with consumers and commanding a price several times higher than ordinary coconuts. Research shows that 2-acetyl-1-pyrroline (2AP) is a key component of the fragrant coconut's aroma, and its content directly affects the intensity of the coconut's aroma. However, current flavor identification of fragrant coconut mainly relies on sensory evaluation and chemical testing. These methods have widespread drawbacks such as high subjectivity, long processing times, and high costs, resulting in significant inefficient production and resource waste in current coconut breeding and production, making it difficult to meet the needs of large-scale breeding and commercial cultivation.

[0003] Therefore, developing a rapid, accurate, and high-throughput molecular marker-based identification technology for highly aromatic perfume coconuts is of great significance for coconut variety breeding, germplasm resource management, and market promotion. Summary of the Invention

[0004] To overcome the technical bottlenecks of existing methods for screening fragrant coconut germplasm, such as low efficiency, long cycle, and high cost, this invention provides an SNP molecular marker for rapid identification of highly fragrant coconut and a high-throughput identification system based on this SNP molecular marker.

[0005] The first aspect of the present invention is to provide an SNP molecular marker for rapid identification of highly aromatic perfume coconut, the sequence of which is shown in SEQ ID NO: 1, wherein the base at the 98th bp position from the 5' end of the sequence shown in SEQ ID NO: 1 is C or T.

[0006] Among them, the fragrance of the heterozygous C / T genotype perfume coconut is significantly stronger than that of the homozygous T / T genotype perfume coconut. Therefore, the C / T genotype perfume coconut can be identified as a highly fragrant perfume coconut, while the homozygous T / T genotype perfume coconut can be identified as a non-highly fragrant perfume coconut.

[0007] A second aspect of the present invention is to provide a primer combination comprising the following primers:

[0008] OUTF: ATGCTATTCAGTTCCTATAATGA,

[0009] OUTR: CAGATACTCGTTAGAGAATGAG,

[0010] P1: TAATGGTGTAGTCAAACTATGAC,

[0011] P2: ATCGACAATTCACCTTGAGGGTA.

[0012] A third aspect of the invention is to provide a kit containing primer combinations as described in the second aspect of the invention.

[0013] A fourth aspect of the invention is to provide the use of SNP markers as described in the first aspect of the invention, primer combinations as described in the second aspect of the invention, or kits as described in the third aspect of the invention in the breeding of highly aromatic coconuts.

[0014] A fifth aspect of the invention is to provide the use of primer combinations as described in the second aspect of the invention or kits as described in the third aspect of the invention in determining the genotype of the SNP markers described in the first aspect of the invention.

[0015] The sixth aspect of the present invention is a method for determining the genotype of the SNP marker described in the first aspect of the present invention, comprising: extracting genomic DNA from a coconut palm to be tested; using the primer combination described in the second aspect of the present invention, performing PCR amplification on the genomic DNA of the coconut palm to be tested to obtain PCR amplification products; performing gel electrophoresis or sequencing on the PCR amplification products; if three bands of 246 bp, 180 bp, and 125 bp are obtained, it is the T / C genotype; if two bands of 246 bp and 125 bp are obtained, it is the T / T genotype.

[0016] The seventh aspect of the present invention is a method for breeding highly aromatic coconuts, wherein the SNP marker described in the first aspect of the present invention is detected in the coconut to be tested, thereby determining whether the coconut to be tested is highly aromatic or not; or the genotype of the SNP marker described in the first aspect of the present invention is determined by the method of the sixth aspect of the present invention, thereby determining whether the coconut to be tested is highly aromatic or not.

[0017] This invention provides a SNP molecular marker for rapid identification of highly aromatic fragrant coconuts, and designs a set of primer combinations for SNP molecular markers that specifically identify highly aromatic fragrant coconut germplasm. By extracting genomic DNA from fragrant coconuts for specific PCR amplification, combined with agarose gel electrophoresis analysis, rapid and accurate molecular identification of highly aromatic and ordinary coconut germplasm can be achieved. Compared with traditional methods, the technical solution of this invention has significant advantages such as simple operation, short detection cycle (identification can be completed at the seedling stage), and objective and reliable results. It provides an efficient technical means for the evaluation of fragrant coconut germplasm resources, molecular marker-assisted breeding, and commercial variety identification, and has great potential application value in the coconut industry. Attached Figure Description

[0018] Figure 1 To design primer combinations based on the SNP markers of this invention.

[0019] Figure 2 The average 2AP content of T / C type and T / T type in 20 samples of fragrant coconut.

[0020] Figure 3 The results of the selection of 20 fragrant coconut varieties. Detailed Implementation

[0021] The present invention will be further described below with reference to the accompanying drawings and specific embodiments to better understand the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0022] Example 1: Obtaining SNP markers related to fragrance

[0023] The test materials consisted of 168 fragrant coconut germplasm accessions, cultivated in the Fragrant Coconut Experimental Garden of the Coconut Research Institute, Chinese Academy of Tropical Agricultural Sciences (19°32′N, 110°46′E). This garden, where the fragrant coconut variety was introduced from Thailand in 1998, is located in a flat area with a tropical monsoon island climate, an average annual temperature of approximately 23.9 ℃, approximately 1953.8 hours of sunshine annually, and approximately 1721.6 mm of annual rainfall.

[0024] Fresh leaves were obtained, and genomic DNA was extracted using the CTAB method and whole-genome resequencing was performed. Variation data for all materials were obtained using GATK software, and the correlation between SNP sites and known agronomic traits was analyzed, identifying 18 candidate SNP sites associated with aroma (Table 1).

[0025] Table 1

[0026] Number Locus ID Genotype OUTF OUTR P1-F P2-R 1 Chr2:46452561 T / G GTGCTATGCTATACCTTAGCTACT TGTCCAATGAAAGAGATATGTTTT ACTTTAAAATAATAATAATAAAAG TTTTATTATTATTATTTTAAAGTTA 2 Chr2:46452564 A / G CTATGCTATACCTTAGCTACTTGA GTCGGTTAAGGAGCCTCAGATAGG CTTTAAAATAATAATAATAAAAGAAG TGCCACATCTTTACTTATTCTTT 3 Chr2:46452570 C / T ACCTTAGCTACTTGACTACCGGGA CTTGAAGTCGGTTAAGGAGCCTCA TAATAATAATAAAAGAAGAAGAAT CAGAATGCCACATCTTTACTTCTG 4 Chr2:46460822 C / T AGGATTAGAAGATGACATGGAGGA AGTCTTAGGCGCCATCTAAGGATGA TCATCCTTAGATGGCGCCTAAGACTT GCACTGCATGGCGCAGCAGGATCTG 5 Chr5:23269364 C / T TATGCATGACTGATGAAAGAGTGC GCACTCTTTCATCAGTCATGCATA CGTCTTGCATTCTTTTTTGAGCAC TAAAAACTAAGGTTGAGGAGCTCCA 6 Chr5:23269389 G / T GGAGCTCCTCAACCTTAGTTTTTA TAAAAACTAAGGTTGAGGAGCTCC GAGCTCCTCAACCTTAGTTTTTAG CAACTACTCGTGAGCCTTCTAGTGA 7 Chr7:10708387 A / G CTTCTTTGAAAGCCACGAACAAGC ACTTGATGTTTGTTACTGGCTTAC CCCCAATCACTGTCTGTGAAACCA AAGAAGGAGCATACTTCTCTCTCC 8 Chr7:27145573 C / T GCCCTGACAACGGCTCTTATATGC TCTGACCGAGGTTGTCTCGGTCGC GAGCAAAGTCGAAGATCGAGCCTC TAGTCGAGGTTGACGTAGACTTTA 9 Chr9:46902528 G / A GACTAAAGATGCTCACTCCTTAAG AGTCCTTTGAAATGGATTACGAGCG TAGCTCCCCAAAGAGGCTAACTCG GTCTTTAAACAAAGGTGGCTAGCT 10 Chr9:48028388 G / C TCCTGACCACTCCTTAAGACCTTC GAGCATTTGTAACTGCAGAGCAAG TATACCAAGGACCCTCCCACCTTG TTACCCTCTATATATGTTGGTTTCG 11 Chr9:48028397 T / C CTCAAGGACGGCCCCAACTGGCCC GTATTCCACGAGCATTTGTAACTG GACCCTCCCACCTTCGAAACCAAT CCGATTCCTTACCCTCTATATATG 12 Chr9:52811515 G / A TCCCCAACAATAAAAATAATACAT TGGGAGGTCTATATCCCCCGGAAA TTCTGCGGTCCGGCAATATCACCG ACAAATCATCATAGGAAGCCGAT 13 Chr10:5057695 C / T CAGCATCAAAGTTCATCATTAAAC TGATGAGTAACATGGCTTGGCTCC GACACATGAGAGTTGCCCCAGTGT TTCTCCTTAAACTCCTATTCAAAG 14 Chr10:5057704 C / G AGTTCATCATTAAACTCTCAAAAG TGAAGGCTATGATGAGTAACATGG GAGTTGCCCCAGTGTTTTGAATAC AGAAGTGTCTTCTCCTTAAACTCC 15 Chr10:39072814 T / C TACCGATGATCTGTCTAGGTATGG TGAGTTGAAGTATTCTTGGTGGAG GCTTTTGAAAAGTTCAAAGAATTC GTTTATTTTTCTACTTCATGTCTA 16 Chr14:25131943 A / G GGAGTCTATCATTGAGGACGGCGC GGATGATCTTCTCTAGCCGTATTC CCATCACCATCCGATAGTGGATCA TATCAGTCTGCTATAGGCAACAGC 17 Chr14:25131946 A / G GTCTATCATTGAGGACGGCGCCGC TTCGGATGATCTTCTCTAGCCGTA TCACCATCCGATAGTGGATCGCTA CACTATCAGTCTGCTATAGGCAAC 18 Chr14:42400719 T / C ATGCTATTCAGTTCCTATAATGA CAGATACTCGTTAGAGAATGAG TAATGGTGTAGTCAAACTATGAC ATCGACAATTCACCTTGAGGGTA

[0027] Further research revealed a site (number 18) capable of aroma identification (Table 2). This site is located at the 98th bp from the 5' end of the sequence shown in SEQ ID NO: 1. In the SEQ ID NO: 1 sequence, this site is indicated by "c," and the base at this site is either c or t. (It should be understood that the use of "c" at the 98th bp from the 5' end of SEQ ID No. 1 in the sequence listing is merely for the normal submission of the nucleotide sequence and does not affect the relevant limitations of claim 1 and the specification of this invention.) Of the 168 germplasm accessions, 40 were of the TC genotype and 128 were of the TT genotype.

[0028] Table 2

[0029]

[0030] The 2AP content of pulp from 168 germplasms was measured. The results showed that the average 2AP content of 128 T / T genotype germplasms was below 5 μg / g, while the average 2AP content of 40 T / C genotype germplasms was above 10 μg / g, with a highly significant difference between the two groups (p < 0.001). This demonstrates that this SNP marker is closely related to coconut aroma; germplasms containing the T / C genotype have a significantly stronger aroma than those containing the T / T genotype. This marker can be used to identify highly aromatic and non-aromatic coconuts, playing a role in molecular-assisted selection.

[0031] Example 2: Breeding method for highly aromatic coconuts based on the above SNP markers

[0032] Target sequence: atgctattcagttcctataatgaacatgctatagccaacatacacctcagggttccatatggctaggagaccaagtaatgg tgtagtcaaactataa[C / T]accctcaaggtgaattgtcgatgcacctcagatcaaagaatcagacacactactacagcatcgagtcagtcattgacgagaaggtagacttcctaatgactactcgaggtagtcacgcttagtgccctcattctctaacgagtatctg

[0033] Primer design: ARMS-PCR primers were designed based on the detection principle that Taq DNA polymerase cannot repair a single base mismatch at the 3' end of the primer, thus hindering amplification. Three PCR products are generated during amplification. The OUTF-OUTR primer pair amplifies the target region as a control band (246 bp), the OUTF-P2 primer pair amplifies the T-type allele band (125 bp), and the P1-OUTR primer pair amplifies the C-type allele band (175 bp). Mismatched bases were intentionally introduced near the 3' end of primers P1 and P2 to reduce the amplification efficiency of non-target sequences and improve primer specificity.

[0034] Primer sequences:

[0035] OUTF: ATGCTATTCAGTTCCTATAATGA

[0036] OUTR: CAGATACTCGTTAGAGAATGAG

[0037] P1: TAATGGTGTAGTCAAACTATGAC

[0038] P2: ATCGACAATTCACCTTGAGGGTA

[0039] ARMS-PCR reaction system: 10 μL of 2*Taq master mix, 0.8 μL of OutF primer, 0.8 μL of OutR primer, 0.4 μL of P1 primer, 0.4 μL of P2 primer, 2 μL of genomic DNA, and ddH2O to bring the system to 20 μL.

[0040] ARMS-PCR reaction steps: ① Pre-denaturation at 95 ℃ for 5 min; ② Denaturation at 95 ℃ for 30 s, annealing at 55 ℃ for 30 s, extension at 72 ℃ for 30 s (40 cycles of amplification); ③ Extension at 72 ℃ for 5 min, then store at 4 ℃ for later use.

[0041] Banding determination: The genotype of the sample was determined by visualizing the band combinations through gel electrophoresis. A 2% agarose gel was prepared, and 5 μL of ARMS-PCR reaction product was used for electrophoresis. Electrophoresis conditions: constant voltage 130 V; current 200 mA; electrophoresis time 25 min. After electrophoresis, the DNA bands were observed under UV light, and photographs were taken using a gel imaging system. C / C homozygotes: 246 bp (control) and 175 bp (C allele) bands appeared; T / T homozygotes: 246 bp (control) and 125 bp (T allele) bands appeared; T / C heterozygotes: three bands appeared: 246 bp, 125 bp, and 175 bp.

[0042] Twenty fragrant coconut samples were randomly selected from 168 samples for verification, including five highly fragrant coconut samples (genotype T / C) and 15 low-fragrance samples (genotype T / T). (The 2AP content of T / C and T / T types in the 20 fragrant coconut samples is shown in Table 3, and the average 2AP content is shown in Table 4.) Figure 2 As shown), the test was performed using the method described above, and the results are as follows. Figure 3 As shown, the primer combination can clearly distinguish between superior and non-superior haplotypes. Five of the 20 samples were superior haplotypes carrying the T / C allele, while 15 were non-superior haplotypes carrying the T / T allele. The TC1-5 germplasm successfully amplified three bands (a 246 bp control band, a 180 bp C allele band, and a 125 bp T allele band), while the TT1-15 germplasm amplified two bands (a 246 bp control band and a 125 bp T allele band). The detection results perfectly matched the expected genotype information, indicating that the genotype detection method of this invention is fully applicable to molecular-assisted selection in the current breeding of highly aromatic coconuts.

[0043] Table 3

[0044]

[0045] The specific embodiments of the present invention have been described in detail above, but they are merely examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. A SNP molecular marker for rapid identification of highly aromatic coconut perfumes, characterized in that, The sequence of the SNP marker is shown in SEQ ID NO: 1, and the base at the 98th bp position from the 5' end of the sequence shown in SEQ ID NO: 1 is C or T.

2. The SNP marker according to claim 1, characterized in that, The fragrance of the heterozygous C / T genotype of perfume coconut, marked by the SNP, was significantly stronger than that of the homozygous T / T genotype of perfume coconut.

3. A primer combination, characterized in that, The primer combination contains the following primers: OUTF: ATGCTATTCAGTTCCTATAATGA, OUTR: CAGATACTCGTTAGAGAATGAG, P1: TAATGGTGTAGTCAAACTATGAC, P2: ATCGACAATTCACCTTGAGGGTA.

4. A kit containing the primer combination as described in claim 3.

5. The use of the SNP marker as described in claim 1 or 2, the primer combination as described in claim 3, or the kit as described in claim 4 in the breeding of highly aromatic coconuts.

6. The use of the primer combination of claim 3 or the kit of claim 4 in determining the genotype of the SNP marker of claim 1 or 2.

7. A method for determining the genotype of the SNP marker according to claim 1 or 2, characterized in that, include: Genomic DNA was extracted from the target fragrant coconut. Using the primer combination described in claim 3, the genomic DNA of the perfume coconut to be tested is amplified by PCR in order to obtain PCR amplification products; The PCR amplification products were subjected to gel electrophoresis or sequencing. If three bands are obtained at 246 bp, 180 bp, and 125 bp, it is the T / C genotype; if two bands are obtained at 246 bp and 125 bp, it is the T / T genotype.

8. A method for breeding highly aromatic coconuts, characterized in that, By detecting the SNP markers as described in claim 1 or 2 on the perfume coconut to be tested, it can be determined whether the perfume coconut to be tested is highly aromatic or not. Alternatively, the genotype of the SNP marker described in claim 1 or 2 can be determined by the method of claim 7, thereby determining whether the perfume coconut to be tested is highly aromatic or not.

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