CN113774159B - Tea tree 4-coumaroyl CoA ligase gene SSR molecular marker primer and application - Google Patents

Tea tree 4-coumaroyl CoA ligase gene SSR molecular marker primer and application Download PDF

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CN113774159B
CN113774159B CN202010515794.0A CN202010515794A CN113774159B CN 113774159 B CN113774159 B CN 113774159B CN 202010515794 A CN202010515794 A CN 202010515794A CN 113774159 B CN113774159 B CN 113774159B
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molecular marker
primer
tea tree
tea
genetic
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CN113774159A (en
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彭靖茹
温立香
檀业维
黄寿辉
张芬
甘志勇
冯春梅
陈家献
袁冬寅
赵媛
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Guangxi Subtropical Crops Research Institute
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Abstract

The invention discloses an SSR molecular marker (SSR 14 CL and SSR15 4 CL) of a tea tree 4-coumaroyl CoA ligase gene and primers thereof, which are researched and developed by the inventor by utilizing a molecular marker technology. Research shows that the SSR primer method is feasible on the whole genome of tea trees according to specific genes, and the obtained primer has high polymorphism, good repeatability, good feasibility and pertinence. Accordingly, the inventors have conducted studies on genetic polymorphisms of tea plant germplasm resources. In conclusion, the research method, the SSR molecular marker and the primer thereof can be widely applied to tea tree variety identification, genetic structure and resource diversity analysis, genetic map construction, functional gene positioning and QTL positioning, molecular marker assisted selection breeding research, and are convenient for further researching tea tree phenolic substance synthesis and tea tree genetic polymorphism and promoting deep development of tea tree resources.

Description

Tea tree 4-coumaroyl CoA ligase gene SSR molecular marker primer and application
Technical Field
The invention belongs to the technical field of molecular markers, and particularly relates to a tea tree 4-coumaroyl CoA ligase gene SSR molecular marker primer and application thereof.
Background
Tea trees are important woody cash crops in China, and tea is the oldest and most popular caffeine-containing beverage in the world. The content of polyphenols in fresh tea leaves is generally 18% -36%, and the polyphenols are main secondary metabolites of tea trees, and have great influence on the formation of tea quality. The composition, content and proportion of polyphenol compounds in fresh leaves, and the conversion degree and form in the tea making process are different, so that the tea has a decisive effect on the formation of tea quality and style, and the color, the aroma and the taste of tea are directly influenced.
In the tea tree phenol synthesis pathway, 4-coumarate acetyl ligase (4-coumaroyl CoA ligase,4 CL) is one of key enzymes, 4-coumarate is mainly catalyzed in a phenylpropane pathway to generate coumaroyl-CoA, the coumaroyl-CoA is catalyzed by chalcone synthase to generate chalcone, and the chalcone is a starting substrate of a tea tree flavonoid pathway and influences the synthesis of polyphenols such as tea tree catechin, anthocyanin and the like. Meanwhile, 4-coumaroyl acid acetyl ligase (4 CL) is also a key enzyme for synthesizing lignin, resveratrol and the like in other crops, and plays an important role in plant growth and development and stress resistance defense reaction. Cloning, expression, bioinformatics analysis and the like of 4CL genes of tea tree, birch, gerbera, amur grape and the like have been carried out. However, research on development and application of SSR molecular markers aiming at tea tree 4CL genes is not reported.
Tea tree morphology is various, and traditional germplasm resource identification is mainly based on phenotype and is easy to influence by environment. Because the molecular marker is not affected by the environment, the variation is only caused by the difference of allele DNA sequences, so the molecular marker has important roles and significance for the research of genetic diversity of tea trees, the identification of high-quality variety resources and the like. The existing molecular marking methods mainly comprise RAPD, RFLP, AFLP, ISSR, SSR, EST-SSR, SRAP, SCoT and the like, the principles of the molecular marking methods are different, marked primers and systems are also different, and certain differences exist in amplified bands and cluster analysis. The simple repeated sequence (simple sequence repeats, SSR) is an important molecular marker, co-dominant inheritance can be applied to a plurality of aspects such as variety identification, genetic map construction, functional gene location and QTL location variety identification, genetic structure and resource diversity analysis, molecular marker assisted breeding selection research and the like.
The high-elevation in 2017 completes the sequencing and assembly of the tea tree assam seed 'cloud anti No. 10' large leaf tea nucleus genome, successfully draws a high-quality genome map of the tea tree, and researches show that 80.9% of the whole genome of the tea tree is a repeated sequence and has very high content, thus indicating that the genome has a large number of SSR molecular markers. The sequenced tea tree genome information is utilized to deeply excavate SSR molecular markers of key enzyme genes of the synthetic pathway of tea phenols, and the development and application of polymorphic primers are obtained, so that a reference basis can be provided for scientific researchers for researching the biochemical and genetic directions of tea trees in the future.
Disclosure of Invention
The invention aims to provide a tea tree 4-coumaroyl CoA ligase gene SSR molecular marker primer and application thereof, so as to further study tea tree phenolic substance synthesis and tea tree genetic polymorphism.
In order to solve the technical problems, the invention adopts the following technical scheme:
the tea tree 4-coumaroyl CoA ligase gene SSR molecular marker has a base sequence of a sequence table SEQ.ID.No.1 or SEQ.ID.No.2, and the marker number is SSR14 CL or SSR15 CL.
The primer for amplifying the SSR molecular marker of the tea tree 4-coumaroyl CoA ligase gene comprises a base sequence with a sequence table SEQ.ID No.3 and a sequence table SEQ.ID No.4, and the primer for amplifying the SSR molecular marker SSR15 4CL comprises a base sequence with a sequence table SEQ.ID No.5 and a sequence table SEQ.ID No. 6.
According to the preparation method of the SSR molecular marker, the specific primer is utilized to carry out PCR amplification on the total DNA of the tea tree, so that a simple repeated sequence is obtained; the primer comprises a base sequence with a sequence table SEQ.ID No.3 and a sequence table SEQ.ID No.4 or a base sequence with a sequence table SEQ.ID No.5 and a sequence table SEQ.ID No. 6.
The preparation method of the SSR molecular marker comprises the following operation steps:
<1> extraction of DNA
Extracting genomic DNA of tea tree by using the kit as a template;
<2> PCR reaction
The reaction system: the total volume of the reaction was 20. Mu.L, wherein 2 XPCR Mix 10. Mu.L, tea tree DNA template 1. Mu.L, ddH 2 O8. Mu.L, 1. Mu.L each of the forward primer and the reverse primer;
the reaction procedure: pre-denaturation at 94 ℃ for 5min, high-temperature denaturation at 94 ℃ for 45s, annealing at 55-60 ℃ for 45s, extension at 72 ℃ for 45s and extension at 72 ℃ for 7min after 35 cycles, and permanent preservation at 4 ℃;
<3> electrophoresis color development
Taking 5 mu L of PCR product, and carrying out constant voltage 200v electrophoresis on 8% non-denaturing polyacrylamide gel for 60min; after the electrophoresis, the gel was developed by staining with silver nitrate.
The forward primer is the primer of the sequence table SEQ.ID No.3 or SEQ.ID No.5, and the reverse primer is the primer of the sequence table SEQ.ID No.4 or SEQ.ID No. 6.
The concentration of the forward primer or the reverse primer of the tea tree DNA template is 10 mu mol/L and is 1-50 ng.
The SSR molecular marker is applied to identification of germplasm resources of tea trees or plants containing phenolic substances, research of genetic diversity, construction of genetic maps, identification of seed purity, genetic relationship of population, evolution or genetic breeding.
The primer of the SSR molecular marker is applied to the aspects of identification of germplasm resources of tea trees or plants containing phenolic substances, research of genetic diversity, construction of genetic maps, identification of seed purity, genetic relationship of population, evolution or genetic breeding.
Aiming at the problem that the research on key enzymes of the phenolic substance synthesis route of the tea tree is not available at present, the inventor utilizes the molecular marker technology to research and develop the SSR molecular marker (SSR 14 4CL and SSR15 4 CL) primer of the tea tree 4-coumaroyl CoA ligase gene and the application thereof on the basis of the public report of the 'cloud anti No. 10' large She Chashu whole genome. Research shows that the SSR primer method is feasible on the whole genome of tea trees according to specific genes, and the obtained primer has high polymorphism, good repeatability, good feasibility and pertinence. Accordingly, the inventors have conducted studies on genetic polymorphisms of tea plant germplasm resources. In conclusion, the research method, the SSR molecular marker and the primer thereof can be widely applied to tea tree variety identification, genetic structure and resource diversity analysis, genetic map construction, functional gene positioning and QTL positioning, seed purity identification, molecular marker assisted selection breeding research, and are convenient for further researching tea tree phenol substance synthesis and tea tree genetic polymorphism and promoting deep development of tea tree resources.
Drawings
FIG. 1 is a polyacrylamide gel electrophoresis of a fragment amplified by a 4CL SSR 6 pair primer of tea tree, wherein: m:2000bp DNA Marker; the tea germplasm for each lane is: 1,6, 12, 18, 24, 30 is green in the xiangbo; 2,7, 13, 19, 25, 31 is Huang Guanyin; 3,8, 14, 20, 26, 32 is early green; 4,9, 15, 21, 27, 33 are fudingding pekoe; 5, 10, 16, 22, 28, 34 are golden buds; 11 17, 23, 29, 35 are rosewood No. 2; the amplified fragments of the corresponding primers of each lane are: 1-5 is 4CL-12SSR primer amplified fragment; 6-11 is 4CL-13SSR primer amplified fragment; 12-17 is 4CL-14SSR primer amplified fragment; 18-23 is 4CL-15SSR primer amplified fragment; 24-29 are 4CL-2SSR primer amplified fragments; 30-35 is 4CL-6SSR primer amplified fragment.
FIG. 2 is a diagram of the polyacrylamide gel electrophoresis of the tea tree 4CL-14SSR primer, in which: m:2000bp DNA Marker; each lane corresponds to the germplasm of the tea as shown in Table 1.
FIG. 3 is a polyacrylamide gel electrophoresis of 4CL-15SSR primers of tea tree, wherein: m:2000bp DNA Marker; each lane corresponds to the germplasm of the tea as shown in Table 1.
Detailed Description
Tea polyphenol synthesis pathway key enzyme 4-coumaroyl CoA ligase (4 CL) SSR molecular marker research
(1) The tea polyphenol synthesis pathway key enzyme 4-coumaroyl CoA ligase (4 CL) gene (GenBank: DQ 194356) mRNA sequence was downloaded from NCBI.
(2) The tea tree 'cloud anti No. 10' whole genome sequence (download address: http:// www.plantkingdomgdb.com/tea_tree/dat) was downloaded. SSR markers are searched in the whole genome of tea trees by using SSR search software, and SSR sequences and sequences with specific lengths (default 100 bp) on the upstream and downstream of the SSR sequences are extracted. Primer3 is used for primer design to obtain primer sequences, SSR repeated motifs and repeated lengths, pre-amplified fragment lengths and the like. 17 pairs of SSR molecular marker primer sequences related to the 4CL genes are prepared by synthesis.
(3) Genomic DNA of 72 tea plant germplasm resources (Table 1) was extracted using a kit HYQspin TM CT Plant DNA Kit produced by Tiangen biosciences, inc.
TABLE 1 sample name of 72 tea leaves
Lanes Sample number and name Lanes Sample number and name Lanes Sample number and name Lanes Sample number and name
1 A27-1 19 Hunan wave green 37 Rogowoad leaf 12 55 Sanjiang No.1
2 A27-2 20 Mei Zhan 38 Rogowoad leaf 17 56 Purple bud No.4
3 Rogowoad leaf fragrance 19 21 Rhododendron simsii (L.) kuntze 39 Board Tang F5 No 57 Sanjiang No.2
4 Rogowoad leaf 21 22 Huang Guanyin 40 Board Tang P number 58 Board Tang S number
5 A91 23 A33 41 Fengping No. 10 59 Jiayou No.2
6 A90 24 A54 42 Golden tea 60 Add You Huangshe
7 A87 25 A51 43 A53 61 Jiayou No.5
8 A20 26 A48 44 White leaf number one 62 Fengping No. 21
9 A73 27 A14 45 Rogowoad leaf 20-1 63 Tetrad red leaf No.6
10 A63 28 Jinguanyin 46 Board Tang F number 64 Liu San sister 1
11 Mei Zhan 29 Bixiang early tea 47 Rogowoad leaf 10-1 65 Board Tang J number
12 A71 30 Big milli of Fuding 48 Wang Fengxin tea garden 66 Gui Gong 2 No.2
13 A85 31 Huang Meigui 49 Fengping No.1 67 Jiayou No.4
14 A76 32 Golden bud 50 Fengping No.5 68 Tetrad red leaf No.4
15 A64 33 Golden peony 51 Lingyun red leaf No.3 69 Jiayou No. 7
16 Jin Xuan 34 Yukylin 52 No. 11 ground No. 48 70 Longlin 42
17 A53 35 Guilu No. 1-2 53 Rogowoad leaf 4 71 Rogowoad leaf 10-2
18 Guilu No. 1-1 36 Rogowoad leaf 2 54 Rogowoad leaf 9 72 Rogowoad leaf 27
(4) From the 17 pairs of SSR primers obtained in the step (2), the obtained molecular marker primers are analyzed for position, type and the like, 2 pairs of primers 4CL-2 and 4CL-6 in 11 pairs of primers located in an intergenic region and 2 pairs of primers 4CL-12 and 4CL-13 in an untranslated region located at the 5' upstream are randomly selected, 6 pairs of primers 4CL-14 and 4CL-15 in an intron region are taken as the total primers (Table 2), and the SSR-PCR reaction is carried out by taking the genomic DNA of the tea plant germplasm resources of 72 parts as templates, wherein the genomic DNA of the tea plant germplasm resources of yellow goddess, rhododendron, golden peony, osmanthus 1, golden buds and green as a total of 6 tea plant germplasm resources is extracted in the step (3), and the PCR amplification products are detected by using a polyacrylamide gel vertical electrophoresis and a rapid silver staining method.
PCR reaction system: the total volume of the reaction was 20. Mu.L, wherein 2 XPCR Mix 10. Mu.L, tea tree DNA template (1-50 ng) 1. Mu.L, ddH 2 O8. Mu.L, forward primer (10. Mu. Mol/L) and reverse primer (10. Mu. Mol/L) were each 1. Mu.L.
PCR reaction procedure: pre-denaturation at 94 ℃ for 5min, high-temperature denaturation at 94 ℃ for 45s, annealing time at 55-60 ℃ for 45s, extension at 72 ℃ for 45s and extension at 72 ℃ for 7min after 35 cycles, and permanent preservation at 4 ℃.
Electrophoresis color development: mu.L of the PCR product was subjected to constant voltage 200v electrophoresis on an 8% non-denaturing polyacrylamide gel for 60min. After the electrophoresis, the gel was developed by staining with silver nitrate. Washing the gel with ddH2O for 3 times, washing for 1min each time, and then adding 0.5-1.5% silver nitrate for dyeing for 10-15 min; then, the mixture was washed with ddH2O 3 times for 1min each. The gel is gently shaken in the developing solution until the gel becomes a developing zoneClear and put into ddH 2 O terminates development. The result of electrophoresis in primer screening is shown in FIG. 1.
The PCR amplification electrophoresis staining result is shown in figure 1, and only two pairs of SSR primers, namely 4CL-14 and 4CL-15, which are positioned in the intron region in 6 pairs of SSR primers have the difference in amplified fragments, and the polymorphism is good and the main band is clear.
Table 2 4-coumaroyl CoA ligase (4 CL) Gene SSR primer
(5) And (3) carrying out PCR amplification reaction by taking 4CL-14 and 4CL-15 as SSR primers and taking 72 parts of tea tree genome DNA extracted in the step (4) as a template to develop the research of genetic polymorphism of tea tree germplasm resources, and obtaining polymorphism map features as shown in figures 2 and 3 (band numbers 11 and 16).
Sequence listing
<110> Guangxi Zhuang autonomous region subtropical crop institute (Guangxi subtropical agricultural product processing institute)
<120> tea tree 4-coumaroyl CoA ligase gene SSR molecular marker primer and application
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 148
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
attttgcact gatttggcaa gagtgtttaa ttttagcatg aattttgatt ttgtttgggt 60
tgggttgggt tgggttgggt tgggtattgt gagtataggg atatgggatg acagaggcag 120
ggccagtatt atcaatgtgc ctggcgtt 148
<210> 2
<211> 135
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
atccgtggtc ctcaaattat gaaaggtact actgctgctg ctgctgctgc tgctgctttc 60
tcttttaata attcattccc tctgctcatc aagttgctca attgtcatca tgcccaacaa 120
atgcaaaaac tattc 135
<210> 3
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
attttgcact gatttggcaa gagt 24
<210> 4
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
aacgccaggc acattgataa tact 24
<210> 5
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
atccgtggtc ctcaaattat gaaa 24
<210> 6
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
gaatagtttt tgcatttgtt gggc 24
<210> 7
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
acgaccattg caattacttc aggt 24
<210> 8
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
caccaagcct tgtccttaga tcat 24
<210> 9
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
ccaattttac gaacacccta tcaa 24
<210> 10
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
tgggttcaaa cctattcaat tcat 24
<210> 11
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
attgccaatt tggttagacg gtta 24
<210> 12
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
gagaatggaa gaggagagga gagc 24
<210> 13
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
tccctctcta tcccctctct gtct 24
<210> 14
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
ccacggacaa cattgttacc ctat 24

Claims (5)

1. The tea tree 4-coumaroyl CoA ligase gene SSR molecular marker is characterized in that the SSR molecular marker sequence is the base sequence of a sequence table SEQ.ID.No.1 or SEQ.ID.No.2.
2. The primer for amplifying the SSR molecular marker of the tea tree 4-coumaroyl CoA ligase gene according to claim 1, wherein the primer sequences of the SSR molecular marker are the base sequences of SEQ ID No.3 and SEQ ID No.4 or the primer sequences of the SSR molecular marker are the base sequences of SEQ ID No.5 and SEQ ID No. 6.
3. The method for preparing the SSR molecular marker as claimed in claim 1, wherein the primer is used for carrying out PCR amplification on the total DNA of tea trees to obtain a simple repeated sequence; the primer sequence is the base sequence of SEQ.ID No.3 and SEQ.ID No.4 or the base sequence of SEQ.ID No.5 and SEQ.ID No. 6.
4. The use of the SSR molecular marker of claim 1 in the identification of germplasm resources of tea trees, research of genetic diversity, construction of genetic maps, identification of seed purity, genetic relationship of populations, evolution or genetic breeding.
5. The application of the primer of the SSR molecular marker in the aspects of identification of germplasm resources of tea trees, research of genetic diversity, construction of genetic maps, identification of seed purity, genetic relationship of population, evolution or genetic breeding.
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Citations (2)

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Publication number Priority date Publication date Assignee Title
CN108998551A (en) * 2018-07-23 2018-12-14 湖南省茶叶研究所（湖南省茶叶检测中心） Tea tree quadruple fluorescence SSR molecular marker detection method
CN110079510A (en) * 2011-04-29 2019-08-02 孟加拉朱特研究所 Encode the polynucleotides of the enzyme from jute Lignin biosynthesis approach

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Publication number Priority date Publication date Assignee Title
US20040010815A1 (en) * 2001-09-26 2004-01-15 Lange B. Markus Identification and characterization of plant genes

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Publication number Priority date Publication date Assignee Title
CN110079510A (en) * 2011-04-29 2019-08-02 孟加拉朱特研究所 Encode the polynucleotides of the enzyme from jute Lignin biosynthesis approach
CN108998551A (en) * 2018-07-23 2018-12-14 湖南省茶叶研究所（湖南省茶叶检测中心） Tea tree quadruple fluorescence SSR molecular marker detection method

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Title
Exploitation of conserved intron scanning as a tool for molecular marker development in the Saccharum complex;Jong-Won Park 等;《Mol Breeding》;第30卷;第987–999页 *
PREDICTED: Camellia sinensis 4-coumarate--CoA ligase 2 (LOC114257011), mRNA;XM_028196719.1;《NCBI》;第1-2页 *
基于SSR分子标记的广西德保县和隆林县 野生古茶树聚类分析;彭靖茹 等;《南方农业学报》;第50卷(第1期);第1-7页 *

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