A set of SNP markers, primer sets and their applications for identifying the germplasm of Nanchuan tea plant population

Abstract

The present invention provides a group of SNP markers for identifying Chongqing large tea tree population germplasm, wherein the population germplasm SNP markers include: the 119192825th position of tea tree Chr8 chromosome is the 301st nucleotide of SEQ ID No.1 in the sequence list, and / or the 40853615th position of tea tree Chr15 chromosome is the 301st nucleotide of SEQ ID No.2 in the sequence list. Using the molecular markers and specific amplification primers of the present invention, it is possible to accurately identify whether the tea tree germplasm is derived from the Chongqing large tea tree population germplasm, and can be used for the traceability identification and protection of the Chongqing large tea tree population germplasm.

Description

Technical Field

[0001] The invention relates to the technical field of biomolecular markers, and in particular to a group of SNP markers for identifying the germplasm of Nanchuan large tea tree population, a primer set and applications. Technical Background

[0002] Tea tree species in taxonomy refers to the genus Camellia in the family Theaceae. Camellia Linn.) Chinese Tea Group (Sec. Thea (L.) Dyer) plant. Tea tree is one of the most important economic crops widely planted in the world and an important source of raw materials for tea drinking and edible applications. However, tea tree production still has problems such as single planting species, serious product homogeneity, and low utilization rate of local specialty germplasm. The effective identification, excavation and utilization of local specialty tea germplasm resources is an important way to solve the problems of tea tree planting and production.

[0003] The Nanchuan tea tree population is distributed at the northern end of the Yunnan-Guizhou Plateau, mainly in the mountainous areas of Nanchuan, Qijiang, Jiangjin, etc. The Nanchuan tea tree population is rich in genetic diversity, and specific or continuous variation traits such as high polyphenols, sweet fragrance, cold resistance, plump buds and leaves, and purple buds have been found. It is a germplasm population with economic value and development potential.

[0004] At present, the industrialization of Nanchuan tea tree germplasm is developing rapidly, and its application is mainly concentrated in Nanchuan District, Chongqing. The area of ​​"Nanchuan tea trees" planted exceeds 10,000 mu, and the developed "Nanchuan big tree tea" has won the title of Chongqing's three major tea brands. However, the further healthy development of Nanchuan tea tree germplasm seedling promotion and product market development still faces the problem of lack of authenticity identification and detection technology. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a set of SNP markers, primer sets and applications for identifying the germplasm of the Nanchuan large tea tree population. The set of SNP markers for the germplasm of the tea tree population provided by the present invention can be used to identify whether the tea tree germplasm is derived from the germplasm of the Nanchuan large tea tree population.

[0006] The present invention provides a group of SNP markers for identifying the germplasm of the Nanchuan large tea tree population, wherein the population germplasm SNP markers include: the 119192825th position of the tea tree Chr8 chromosome is the 301st nucleotide of SEQ ID No.1 in the sequence list, and / or the 40853615th position of the tea tree Chr15 chromosome is the 301st nucleotide of SEQ ID No.2 in the sequence list.

[0007] The present invention also provides a primer set for amplifying the above-mentioned SNP marker for identifying the germplasm of the Nanchuan large tea plant population. The 119192825th position of the tea plant Chr8 chromosome is amplified and identified by the nucleotide sequence of primer set 1 as shown in SEQ ID No. 3-5.

[0008] The tea plant Chr15 chromosome position 40853615 is amplified and identified by the nucleotide sequence of primer set 2 as shown in SEQ ID No. 6-8.

[0009] The present invention also provides the use of the above SNP marker in identifying whether the tea tree germplasm is derived from the Nanchuan large tea tree population germplasm.

[0010] Further, the application of the above SNP markers in identifying the germplasm of the Nanchuan large tea tree population includes the following steps:

[0011] 1) Extracting DNA from tea plant materials;

[0012] 2) using the extracted DNA as a template, and performing a PCR amplification reaction using the primer set shown in SEQ ID No. 3 to 5;

[0013] 3) Obtain the fluorescence signal for genotyping. When the fluorescence signal genotyping at position 119192825 of the tea tree Chr8 chromosome is CC, it is the germplasm source of the Nanchuan large tea tree population. When it is TT, it is the tea tree germplasm from other sources.

[0014] Alternatively, include the following steps:

[0015] 1) Extracting DNA from tea plant materials;

[0016] 2) using the extracted DNA as a template, and performing a PCR amplification reaction using the primer set shown in SEQ ID No. 6 to 8;

[0017] 3) Obtain the fluorescence signal for genotyping. When the fluorescence signal genotyping of the tea tree material Chr15 chromosome site 40853615 is TT, it is the germplasm source of the Nanchuan large tea tree population. When it is CC, it is the tea tree germplasm from other sources.

[0018] Alternatively, include the following steps:

[0019] 1) Extracting DNA from tea plant materials;

[0020] 2) using the extracted DNA as a template, and performing a PCR amplification reaction using the primer sets shown in SEQ ID Nos. 3 to 5 and SEQ ID Nos. 6 to 8;

[0021] 3) Obtain the fluorescence signal for genotyping. When the fluorescence signal genotype at position 119192825 of the tea tree Chr8 chromosome is CC, it is the germplasm source of the Nanchuan large tea tree population; when it is TT, it is the germplasm source of tea trees from other sources; and when the fluorescence signal genotype at position 40853615 of the tea tree material Chr15 chromosome is TT, it is the germplasm source of the Nanchuan large tea tree population; when it is CC, it is the germplasm source of tea trees from other sources.

[0022] Furthermore, the total reaction volume of PCR in step 2) is 10 μL, including 2.5 μL DNA (concentration is about 10 ng / μl), 5 μL GUDE FLu-Arms 2x PCR Mix, 0.5 μL mixed primers and 2 μL water.

[0023] Furthermore, the PCR amplification reaction conditions in step 2) are as follows: 95°C for 10 min; 95°C for 20 s, 61-55°C for 1 min for 10 cycles; 95°C for 20 s, 55°C for 1 min, 28-45 cycles, 25°C for 30 s.

[0024] Furthermore, the PCR amplification reaction in step 2) is performed on CFX Connect TM The results were performed on a Real-Time System (BIO-RAD).

[0025] The present invention provides a group of SNP markers for identifying the germplasm of Nanchuan tea tree population. The 119192825th position of tea tree Chr8 chromosome is the 301st nucleotide of SEQ ID No.1 in the sequence list; the 40853615th position of tea tree Chr15 chromosome is the 301st nucleotide of SEQ ID No.2 in the sequence list.

[0026] The present invention also provides a primer set for amplifying the SNP markers for identifying the germplasm of the Nanchuan tea tree population described in the above scheme. Primer set 1: amplifying the nucleotide sequence of the 119192825th position of the tea tree Chr8 chromosome as shown in SEQ ID No.3 to 5. Primer set 2: amplifying the nucleotide sequence of the 40853615th position of the tea tree Chr15 chromosome as shown in SEQ ID No.6 to 8.

[0027] The above-mentioned tea tree chromosome number and SNP marker site provided by the present invention refer to the latest updated data of the Tea Plant Information Archive (TPIA) database in 2020. In specific applications, if the reference tea tree genome is adjusted, it should be aligned with the reference genome data in the present invention.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1. The present invention provides a set of SNP markers, primer sets and applications for identifying the germplasm of the Nanchuan large tea tree population, which fills the gap in the authenticity identification technology of the Nanchuan large tea tree population germplasm, can be used for the traceability identification and protection of the Nanchuan large tea tree population germplasm, and is of great significance for promoting the healthy development of the industrial utilization of the Nanchuan large tea tree population germplasm.

[0030] 2. The molecular marker SNP polymorphism site development and detection technology has the characteristics of high accuracy and good repeatability in the molecular-assisted breeding germplasm typing and germplasm resource identification. The present invention uses simplified genome sequencing technology to find that the Nanchuan tea tree germplasm belongs to a population that is relatively independent of other surveyed germplasms, which provides a theoretical basis for the development of SNP marker sites for identifying whether tea tree germplasm is derived from the Nanchuan tea tree population germplasm. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Example 1: Using tea tree molecular marker Chr8 chromosome 119192825 site to detect 20 tea tree resource typing maps;

[0032] Among them: RFU for Allele 1-FAM refers to: relative fluorescence unit of FAM fluorescent group

[0033] RFU for Allele 2-VIC means: Relative Fluorescence Unit of VIC Fluorophore

[0034] The square in the figure indicates: TT genotype; the triangle indicates: CT genotype; the circle indicates: CC genotype; the cross indicates: no signal detected;

[0035] Figure 2 Example 2: Using tea tree molecular marker Chr15 chromosome 40853615 site to detect 20 tea tree resource typing maps;

[0036] Among them: RFU for Allele 1-FAM refers to: relative fluorescence unit of FAM fluorescent group

[0037] RFU for Allele 2-VIC means: Relative Fluorescence Unit of VIC Fluorophore

[0038] In the figure, the square represents CC genotype; the triangle represents CT genotype; the circle represents TT genotype; the cross represents no signal detected. DETAILED DESCRIPTION

[0039] The purpose, technical scheme and beneficial effects of the present invention are further described below in conjunction with the embodiments. The described embodiments are helpful for those skilled in the art to better understand the present invention and do not constitute a limitation of the present invention.

[0040] Example 1

[0041] 1. Plant materials and their genomic DNA extraction

[0042] Ten Nanchuan tea plant germplasms and 10 tea plant germplasms from other sources were selected as plant materials (Table 1). Tender leaf tissues of each plant were picked and then directly frozen in liquid nitrogen. DNA samples were extracted using the Tiangen DNA extraction kit, and the integrity of DNA was detected by 1.0% agarose gel electrophoresis. The DNA concentration, nucleotide OD260 / 280 and OD260 / 230 quality values ​​were detected using a NanoDrop spectrophotometer.

[0043] 2. PCR amplification reaction system and conditions

[0044] PCR amplification reaction system (10 μL): 2.5 μL DNA (concentration of about 10 ng / μl), 5 μL GUDE FLu-Arms 2x PCR Mix, 0.5 μL mixed primers and 2 μL water. PCR reaction conditions: 95 ℃ 10 min; 95 ℃ 20 s, 61-55 ℃ 1 min 10 cycles; 95 ℃ 20 s, 55 ℃ 1 min, 28-45 cycles; 25 ℃ 30 s. The amplification primers used were primer set 1, and its nucleotide sequence is shown in SEQ ID No. 3-5, that is, the 119192825 polymorphic site of tea plant Chr8 chromosome was amplified. PCR amplification reaction was performed on CFX Connect TM The results were performed on a Real-Time System (BIO-RAD).

[0045] 3. Fluorescence signal and genotyping

[0046] Leverage CFX Connect TM Real-Time System (BIO-RAD) fluorescence signal was used to genotype 20 tea tree materials. When primer set 1 was used to amplify the 119192825th site of tea tree Chr8 chromosome, when the fluorescence signal genotyping was CC, it was the germplasm source of the Nanchuan large tea tree population, and when the site was TT, it was the tea tree germplasm from other sources (Table 1).

[0047] Table 1. Genotyping of 20 tea plant resource materials using primer set 1

[0048]

[0049] Note: The expression '*' means that no signal was detected.

[0050] Example 2

[0051] 1. Plant materials and their genomic DNA extraction

[0052] Ten Nanchuan tea plant germplasms and 10 tea plant germplasms from other sources were selected as plant materials (Table 2). Tender leaf tissues of each plant were picked and then directly frozen in liquid nitrogen. DNA samples were extracted using the Tiangen DNA extraction kit, and the integrity of DNA was detected by 1.0% agarose gel electrophoresis. The DNA concentration, nucleotide OD260 / 280 and OD260 / 230 quality values ​​were detected using a NanoDrop spectrophotometer.

[0053] 3. PCR amplification reaction system and conditions

[0054] PCR amplification reaction system (10 μL): 2.5 μL DNA (concentration of about 10 ng / μl), 5 μL GUDE FLu-Arms 2x PCR Mix, 0.5 μL mixed primers and 2 μL water. PCR reaction conditions: 95 ℃ 10 min; 95 ℃ 20 s, 61-55 ℃ 1 min 10 cycles; 95 ℃ 20 s, 55 ℃ 1 min, 28-45 cycles; 25 ℃ 30 s. The amplification primers used were primer set 2, and the amplified nucleotide sequences were shown in SEQ ID No. 6-8, that is, the polymorphic site 40853615 of tea plant Chr15 chromosome was amplified. PCR amplification reaction was performed on CFX Connect TM The results were performed on a Real-Time System (BIO-RAD).

[0055] 4. Fluorescence signal and genotyping

[0056] Leverage CFX Connect TM Real-Time System (BIO-RAD) fluorescence signal was used to genotype 20 tea tree materials. When the 40853615th position of tea tree chromosome Chr15 was amplified using primer set 2, when the fluorescence signal genotyping was TT, it was the germplasm source of the Nanchuan large tea tree population, and when the site was CC, it was the tea tree germplasm from other sources (Table 2).

[0057] Table 2. Genotyping of 20 tea plant resource materials using primer set 2

[0058]

[0059] Note: The expression '*' means that no signal was detected.

[0060] Although some of the genotypes in Table 1 and Table 2 are heterozygous (CT or TC), their proportion is relatively small (5%). At the same time, considering that there may be a small amount of natural distant hybridization in the germplasm of the Nanchuan large tea tree population, it is still proved that the present invention can be used for the identification of the traceability of the germplasm of the Nanchuan large tea tree population. In specific implementation, the genotyping results of primer set 1 and primer set 2 can also be combined for comprehensive judgment.

[0061] The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any modification, replacement, etc. within the scope of the claims of the present invention shall be deemed to be equivalent to the implementation case and shall fall within the protection scope of the technical solution of the present invention. SEQUENCE LISTING <110> Southwest University <120> A set of SNP markers, primer sets and their applications for identifying the germplasm of Nanchuan tea plant population <130> Tea Plant Information Archive (TPIA) <160> 8 <170> PatentIn version 3.5 <210> 1 <211> 601 <212> DNA <213> Camellia sinensis <400> 1 tattttcaat gaaattcttg ccaagatgaa aactagattg tcaggttagt aagctaggtc 60 cttcaatttg gttggtagaa ctactcctat acagtcaatc acctctgtca tccccactta 120 taacatgtaa tccatggaac ttcctaggaa aatctgtgat gaaattaata aaattaaccg 180 caatttcctt tgggatggta ctgtgaccca taagaaagtc catttggtca attgggatag 240 tgtgtataga agcaaagctg agggagggct gggcattaga aaggctagag accaaaatgc 300 yactctgcta accaaacttg ggtggaatt tttgagtgat tagged tttggcgtag agtgcttcat tccaaatact tgaaacataa caacttctat tcttggccaa tgaagaagag gtcctcacat atctggaga gcatgtgtac atatagaga gtcctcaga agggagtcaa atggactgtg ggtaatgaga aggatatctc tctctgaaaa gattgatggt gtggtaacca tcctctttac caggttaata acacctatcc tattaatgat tttgataagg tctaacttaa t 601 <210> 2 <211> 601 <212> DNA <213> Camellia sinensis <400> 2 gtatgttttt caatgacat tccacttttg cttccatatt tctaggaaaa accacgcaat agaaccattt gattttacga father aatactgtt tttgatcca aatcacacat 180. aggatactgt ttgtattaga tgcaagccta gccataccta acaaccgaac taaaacttta cgccatgctt ttagtctcaa caagtcttag taccttttgc ttatcttagc caagctagtg aaccgtgcta ggtttgcgca atgccgcact agaaagcctt ctccactgac 300 ygagctacaa cttggggcta aaccaactct cttggtcaaa aaccgaacta agaatgctgc 360 cccaaatgct caaccgggct aaagaccctt gctcaaacag ggctaagaat ggatagtatc 420 ccacgtagcc gagctaggat ttcatttatc ttgcctttgt ttcttttagc acggtccaac 480 gatctgtgcg taacaccttt ctttttcact ttgtagactt tgactaggtt gcccaggcac 540 tcctttgaaa gtccaaccga gataaccaac gtcatctcag caccgagctg gtactctgct 600 a 601 <210> 3 <211> 45 <212> DNA <213> Artificial Sequence <400> 3 gaaggtgacc aagttcatgc tagaaaggct agagaccaaa atgct 45 <210> 4 <211> 45 <212> DNA <213> Artificial Sequence <400> 4 gaaggtcgga gtcaacggat tagaaaggct agagaccaaa atgcc 45 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <400> 5 ggaatgaagc actctacgcc aa 22 <210> 6 <211> 46 <212> DNA <213> Artificial Sequence <400> 6 gaaggtgacc aagttcatgc tcactagaaa gccttctcca ctgacc 46 <210> 7 <211> 46 <212> DNA <213> Artificial Sequence <400> 7 gaaggtcgga gtcaacggat tcactagaaa gccttctcca ctgact 46 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <400> 8 agcattctta gttcggtttt tgacc 25

Claims

1. A set of SNP markers for identifying the germplasm of the Nanchuan tea plant population, characterized in that: The population germplasm SNP marker includes SEQ ID No.1 and / or SEQ ID No.2, wherein the 301st nucleotide of SEQ ID No.1 is C or T, and the 301st nucleotide of SEQ ID No.2 is T or C; The nucleotide at position 301 of SEQ ID No. 1 is amplified and identified by the nucleotide sequence of primer set 1 as shown in SEQ ID Nos. 3 to 5; The 301st nucleotide of SEQ ID No. 2 is amplified and identified by the nucleotide sequence of primer set 2 as shown in SEQ ID Nos. 6 to 8.

2. Use of the SNP marker described in claim 1 in identifying whether tea germplasm is derived from the Nanchuan large tea population germplasm.

3. The use of the SNP marker according to claim 2 in identifying the germplasm of the Nanchuan large tea plant population, characterized in that: The following steps are involved: 1) Extracting DNA from tea plant materials; 2) using the extracted DNA as a template, and performing a PCR amplification reaction using the primer set shown in SEQ ID No. 3 to 5; 3) Obtaining the fluorescence signal for genotyping, when the 301st nucleotide fluorescence signal genotyping of SEQ ID No.1 is CC, it is from the Nanchuan large tea tree population germplasm, and when it is TT, it is from other sources of tea tree germplasm.

4. The use of the SNP marker according to claim 2 in identifying the germplasm of the Nanchuan large tea plant population, characterized in that: The following steps are involved: 1) Extracting DNA from tea plant materials; 2) using the extracted DNA as a template, and performing a PCR amplification reaction using the primer set shown in SEQ ID No. 6 to 8; 3) Obtaining the fluorescence signal for genotyping, when the 301st nucleotide fluorescence signal genotyping of SEQ ID No. 2 is TT, it is from the Nanchuan large tea tree population germplasm, and when it is CC, it is from other sources of tea tree germplasm.

5. The use of the SNP marker according to claim 2 in identifying the germplasm of the Nanchuan large tea plant population, characterized in that: The following steps are involved: 1) Extracting DNA from tea plant materials; 2) using the extracted DNA as a template, and performing a PCR amplification reaction using the primer sets shown in SEQ ID Nos. 3 to 5 and SEQ ID Nos. 6 to 8; 3) Obtaining fluorescence signals for genotyping, when the 301st nucleotide fluorescence signal genotyping of SEQ ID No.1 is CC, it is the germplasm source of the Nanchuan large tea tree population, and when it is TT, it is the tea tree germplasm from other sources; and when the 301st nucleotide fluorescence signal genotyping of SEQ ID No.2 is TT, it is the germplasm source of the Nanchuan large tea tree population, and when it is CC, it is the tea tree germplasm from other sources.

6. The use of the SNP marker according to any one of claims 3 to 5 in identifying the germplasm of the Nanchuan large tea plant population, characterized in that: The total reaction volume of PCR in step 2) is 10 μL, including 2.5 μL DNA, 5 μL GUDE FLu-Arms 2x PCRMix, 0.5 μL mixed primers and 2 μL water.

7. The use of the SNP marker according to any one of claims 3 to 5 in identifying the germplasm of the Nanchuan large tea plant population, characterized in that: The PCR amplification reaction conditions of step 2) are as follows: 95°C for 10 min; 95°C for 20 s, 61-55°C for 1 min for 10 cycles; 95°C for 20 s, 55°C for 1 min, 28-45 cycles; 25°C for 30 s.

8. The use of the SNP marker according to any one of claims 3 to 5 in identifying the germplasm of the Nanchuan large tea plant population, characterized in that: Step 2) PCR amplification reaction in CFX Connect TM The experiment was carried out on a Real-Time System instrument.

Images