阅读说明：本技术 茶树4-香豆酰CoA连接酶基因SSR分子标记引物及应用 (SSR molecular marker primer of tea tree 4-coumaroyl CoA ligase gene and application ) 是由 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 于 2020-06-09 设计创作，主要内容包括：本发明公开了发明人利用分子标记技术研究开发了茶树4-香豆酰CoA连接酶基因SSR分子标记(SSR14 4CL和SSR15 4CL)及其引物。研究表明,本发明依据具体特定的基因,在茶树全基因组上开发其SSR引物方法可行,所得引物多态性高、重复性好,具有较好的可行性、针对性。据此,发明人开展了茶树种质资源遗传多态性的研究。综上,本发明的研究方法、SSR分子标记及其引物,可广泛应用于茶树品种鉴定、遗传结构和资源多样性分析、遗传图谱构建,功能基因定位及QTL定位、分子标记辅助选育种研究中,便于进一步研究茶树酚类物质合成以及茶树遗传多态性,促进深入开发茶树资源。(The invention discloses SSR molecular markers (SSR 144 CL and SSR 154 CL) of tea tree 4-coumaroyl CoA ligase genes and primers thereof, which are researched and developed by an inventor by utilizing a molecular marker technology. Research shows that the method for developing the SSR primer on the whole genome of the tea trees is feasible according to specific genes, and the obtained primer has high polymorphism, good repeatability, and good feasibility and pertinence. Accordingly, the inventors have conducted studies on genetic polymorphisms of tea plant germplasm resources. In conclusion, the research method, SSR molecular marker and primers thereof can be widely applied to variety identification of tea trees, analysis of genetic structure and resource diversity, construction of genetic maps, functional gene positioning and QTL positioning, and molecular marker-assisted breeding selection research, are convenient for further research on synthesis of phenolic substances of tea trees and genetic polymorphism of tea trees, and promote deep development of tea tree resources.)

1. The tea tree 4-coumaroyl CoA ligase gene SSR molecular marker is characterized by having a base sequence of a sequence table SEQ.ID.No.1 or SEQ.ID.No.2, and the marker number is SSR 144 CL or SSR 154 CL.

2. The primer for amplifying the tea tree 4-coumaroyl CoA ligase gene SSR molecular marker in claim 1, which is characterized in that the primer for the molecular marker SSR 144 CL comprises the base sequences of the sequence tables SEQ ID No.3 and SEQ ID No.4, and the primer for the molecular marker SSR 154 CL comprises the base sequences of the sequence tables SEQ ID No.5 and SEQ ID No. 6.

3. A method for producing SSR molecular markers according to claim 1 characterized by: carrying out PCR amplification on the total DNA of the tea trees by using a specific primer to obtain a simple repetitive sequence; the primer comprises base sequences of sequence tables SEQ ID No.3 and SEQ ID No.4 or sequence tables SEQ ID No.5 and SEQ ID No. 6.

4. A method for the preparation of SSR molecular markers according to claim 3 characterized by the following operating steps:

<1> extraction of DNA

Extracting the genomic DNA of the tea tree by using the kit as a template;

<2> PCR reaction

Reaction system: the total reaction volume was 20. mu.L, 2 XPCR Mix 10. mu.L, tea tree DNA template 1. mu.L, ddH₂O8. mu.L, each of the forward primer and the reverse primer1μL；

Reaction procedure: pre-denaturation at 94 ℃ for 5min, performing high-temperature denaturation at 94 ℃ for 45s in each cycle, annealing at 55-60 ℃ for 45s, extending at 72 ℃ for 7min after 35 cycles, and permanently storing at 4 ℃;

<3> electrophoretic color development

Taking 5 mu L of PCR product, and carrying out electrophoresis on 8% non-denaturing polyacrylamide gel at constant voltage of 200v for 60 min; after electrophoresis, the gel was stained with silver nitrate and developed.

5. A method of making a SSR molecular marker according to claim 4 characterized in that: the forward primer is a primer of a sequence table SEQ ID No.3 or SEQ ID No.5, and the reverse primer is a primer of a sequence table SEQ ID No.4 or SEQ ID No. 6.

6. A method of making a SSR molecular marker according to claim 4 characterized in that: the tea tree DNA template is 1-50 ng, and the concentration of the forward primer or the reverse primer is 10 mu mol/L.

7. The SSR molecular marker of claim 1 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 and evolution of populations or genetic breeding.

8. The SSR molecular marker primer of claim 2 is applied to identification of tea plant or plant germplasm resources containing phenolic substances, research of genetic diversity, construction of genetic maps, identification of seed purity, genetic relationship and evolution of populations or genetic breeding.

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 tree is an important woody economic crop in China, and tea is the oldest and most popular beverage containing caffeine in the world. The content of polyphenols in fresh tea leaves is generally between 18% and 36%, and the polyphenols are main secondary metabolites of tea trees and have great influence on the quality of the tea leaves. 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 formation of the quality and style of tea leaves is decisive, and the color, aroma and taste of the tea leaves are directly influenced.

In the synthesis pathway of tea tree phenols, 4-coumarate acetyl ligase (4-coumaroyl CoA ligase, 4CL) is one of key enzymes, 4-coumarate is mainly catalyzed in a phenylpropane pathway to generate coumaroyl coenzyme A, the coumaroyl coenzyme A generates chalcone under the catalysis of chalcone synthetase, and the chalcone is an initial substrate of a tea tree flavonoid pathway and influences the synthesis of polyphenol substances such as tea tree catechins and anthocyanin and the like. Meanwhile, 4-coumaroyl acid acetyl ligase (4CL) 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-resistant defense reaction. The 4CL genes of tea trees, birch trees, gerbera jamesonii, vitis amurensis and the like have been cloned and expressed, subjected to bioinformatics analysis and the like. However, the development and application research of SSR molecular markers aiming at the tea tree 4CL gene is not reported.

Tea trees are various in shapes, and traditional germplasm resources are mainly identified on the basis of phenotypes and are easily influenced by the environment. Because the molecular marker is not influenced by the environment, and the variation only comes from the difference of allele DNA sequences, the molecular marker has important functions and significance for the research of genetic diversity of tea trees, the identification of high-quality variety resources and the like. At present, 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 different, and amplified bands and clustering analysis have certain differences. The Simple Sequence Repeats (SSR) is an important molecular marker, is co-dominant heredity, and can be applied to various aspects such as variety identification, genetic map construction, functional gene mapping and QTL mapping variety identification, genetic structure and resource diversity analysis, molecular marker-assisted breeding research and the like.

High-standing will in 2017 completes sequencing and assembly of the Assamum 'Yunzi No. 10' Camellia sinensis nuclear genome of the tea tree, 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 repetitive sequence and has very high content, which indicates that the tea tree has a large amount of SSR molecular markers. By utilizing the sequenced tea tree genome information, key enzyme gene SSR molecular markers in the tea tree phenolic substance synthesis pathway are deeply excavated, and polymorphic primers are obtained for development and application, so that a reference basis can be provided for researchers researching tea biochemistry and tea tree gene direction in the future.

Disclosure of Invention

The invention aims to solve the technical problem of providing tea tree 4-coumaroyl CoA ligase gene SSR molecular marker primers and application thereof so as to further research the synthesis of tea tree phenolic substances 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 is marked with SSR 144 CL or SSR 154 CL.

The primer for amplifying the tea tree 4-coumaroyl CoA ligase gene SSR molecular marker comprises a base sequence with a sequence table SEQ ID No.3 and a base sequence with a sequence table SEQ ID No.4, and the primer for molecular marker SSR 154 CL 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, a specific primer is used for carrying out PCR amplification on total DNA of the tea tree to obtain a simple repetitive sequence; the primer comprises base sequences of sequence tables SEQ ID No.3 and SEQ ID No.4 or sequence tables SEQ ID No.5 and SEQ ID No. 6.

The preparation method of the SSR molecular marker comprises the following operation steps:

<1> extraction of DNA

Extracting the genomic DNA of the tea tree by using the kit as a template;

<2> PCR reaction

Reaction system: the total reaction volume was 20. mu.L, 2 XPCR Mix 10. mu.L, tea tree DNA template 1. mu.L, ddH₂O8. mu.L, 1. mu.L each of the forward primer and the reverse primer;

reaction procedure: pre-denaturation at 94 ℃ for 5min, performing high-temperature denaturation at 94 ℃ for 45s in each cycle, annealing at 55-60 ℃ for 45s, extending at 72 ℃ for 7min after 35 cycles, and permanently storing at 4 ℃;

<3> electrophoretic color development

Taking 5 mu L of PCR product, and carrying out electrophoresis on 8% non-denaturing polyacrylamide gel at constant voltage of 200v for 60 min; after electrophoresis, the gel was stained with silver nitrate and developed.

The forward primer is a primer of a sequence table SEQ ID No.3 or SEQ ID No.5, and the reverse primer is a primer of a sequence table SEQ ID No.4 or SEQ ID No. 6.

The DNA template of the tea tree is 1-50 ng, and the concentration of the forward primer or the reverse primer is 10 mu mol/L.

The SSR molecular marker is applied to identification of tea tree or plant germplasm resources containing phenolic substances, research on genetic diversity, construction of genetic maps, identification of seed purity, genetic relationship and evolution of populations or genetic breeding.

The SSR molecular marker primer is applied to identification of tea tree or plant germplasm resources containing phenolic substances, research of genetic diversity, construction of genetic maps, identification of seed purity, genetic relationship and evolution of populations or genetic breeding.

Aiming at the problem that key enzyme research of the phenolic substance synthesis pathway of tea trees is still few at present, the inventor researches and develops the primers of the tea tree 4-coumaroyl CoA ligase gene SSR molecular markers (SSR 144 CL and SSR 154 CL) and the application thereof by using a molecular marker technology on the basis of the public report of 'Yuntan No. 10' large-leaf tea tree whole genome. Research shows that the method for developing the SSR primer on the whole genome of the tea trees is feasible according to specific genes, and the obtained primer has high polymorphism, good repeatability, and good feasibility and pertinence. Accordingly, the inventors have conducted studies on genetic polymorphisms of tea plant germplasm resources. In conclusion, the research method, SSR molecular marker and primers thereof can be widely applied to the researches of tea tree variety identification, genetic structure and resource diversity analysis, genetic map construction, functional gene positioning and QTL positioning, seed purity identification and molecular marker-assisted seed selection, are convenient for further researching tea tree phenolic substance synthesis and tea tree genetic polymorphism, and promote the deep development of tea tree resources.

Drawings

FIG. 1 is a polyacrylamide gel electrophoresis diagram of amplified fragments of 4CL SSR 6 pairs of primers of tea trees, wherein: m: 2000bp DNA Marker; the corresponding tea germplasm of each lane is as follows: 1, 6, 12, 18, 24, 30 is Hunan green; 2, 7, 13, 19, 25, 31 is majoram; 3, 8, 14, 20, 26, 32 is Bixiangzao; 4, 9, 15, 21, 27, 33 are fuding damao; 5, 10, 16, 22, 28, 34 is golden bud; 11, 17, 23, 29, 35 is luoxiang No. 2; the primer amplified fragments corresponding to each lane are: 1-5 are 4CL-12SSR primer amplification fragments; 6-11 are 4CL-13SSR primer amplification fragments; 12-17 are 4CL-14SSR primer amplification fragment; 18-23 are 4CL-15SSR primer amplification fragments; 24-29 are 4CL-2SSR primer amplification fragments; 30-35 is a 4CL-6SSR primer amplification fragment.

FIG. 2 is an electrophoretogram of 4CL-14SSR primer polyacrylamide gel of tea tree, in which: m: 2000bp DNA Marker; the tea germplasm for each lane is shown in Table 1.

FIG. 3 is an electrophoretogram of 4CL-15SSR primer polyacrylamide gel of tea tree, in which: m: 2000bp DNA Marker; the tea germplasm for each lane is shown in Table 1.

Detailed Description

SSR molecular marker research of key enzyme 4-coumaroyl CoA ligase (4CL) in tea polyphenol synthesis pathway of tea trees

(1) The mRNA sequence of the key enzyme 4-coumaroyl CoA ligase (4CL) gene (GenBank: DQ194356) of the tea tree tea polyphenol synthesis pathway is downloaded from NCBI.

(2) The tea tree 'Yunkan No. 10' whole genome sequence (download address: http:// www.plantkingdomgdb.com/tea _ tree/dat) was downloaded. An SSR marker of the tea tree is searched in the whole genome of the tea tree by using SSR search software, and an SSR sequence and a sequence with a specific length (default 100bp) of the upstream and the downstream of the SSR sequence are extracted. Primer3 is used for primer design to obtain primer sequence, SSR repeated element and repeated length, pre-amplified fragment length, etc. According to the synthesis, 17 pairs of SSR molecular marker primer sequences related to the 4CL gene are prepared.

(3) Genomic DNA of 72 tea Plant germplasm resources (Table 1) was extracted using HYQspin TM CT Plant DNA Kit, a Kit produced by Tiangen biology, Inc.

TABLE 172 tea sample names

Lane lane	Sample number and name	Lane lane	Sample number and name	Lane lane	Sample number and name	Lane lane	Sample number and name
								1	A27-1	19	Xiangbo green	37	Luoxiang 12	55	Sanjiang No.1
2	A27-2	20	Plum blossom	38	Luoxiang 17	56	Number 4 Diziya
								3	Luoxiang 19	21	Zijuan (Azalea Arcuata Batsch.)	39	Bantang F5 number	57	Sanjiang No.2
4	Luoxiang 21	22	Yellow kwan-yin	40	Tang P board	58	Tang Dynasty S-shaped board
								5	A91	23	A33	41	Fenxiangping No. 10	59	Jiayou No.2
6	A90	24	A54	42	Golden tea	60	Folium Et cacumen Alstoniae Yunnanensis
								7	A87	25	A51	43	A53	61	Jiayou No.5
8	A20	26	A48	44	Root of white leaf-Hayao	62	Wind phasePing 21 #
								9	A73	27	A14	45	Luoxiang 20-1	63	Tetragenous leaf No.6
10	A63	28	Golden kwan-yin	46	Tang Dynasty No. F	64	Liu Sanjie No.1
								11	Plum blossom	29	Bixiangzao tea	47	Luoxiang 10-1	65	Tang J board
12	A71	30	Lucky tripod big hair	48	Wangfeng new tea garden	66	Guihong No.2
								13	A85	31	Huang Meigui	49	Fengyiping No.1	67	Jiayou No.4
14	A76	32	Golden bud	50	Fengyiping No.5	68	Tetragenous red leaf No.4
								15	A64	33	Golden peony	51	Lingyun Hongye No.3	69	Jiayou No. 7
16	Jin Xuan	34	Jade kylin	52	Number 11, number 48	70	Longlin 42
								17	A53	35	Guilv No. 1-2	53	Luoxiang 4	71	Luoxiang 10-2
18	Guilv No. 1-1	36	Luoxiang 2	54	Luoxiang 9	72	Luoxiang 27

(4) Analyzing the position, type and the like of the obtained molecular marker primers from 17 pairs of SSR primers obtained in the step (2), randomly selecting 2 pairs of primers 4CL-2 and 4CL-6 from 11 pairs of primers positioned in an intergenic region, 2 pairs of primers 4CL-12 and 4CL-13 positioned in a 5' upstream untranslated region, and 6 pairs of primers 4CL-14 and 4CL-15 positioned in an intron region (Table 2), carrying out PCR (polymerase chain reaction) by taking total 6 tea plant germplasm resource genome DNAs of yellow kwan-yin, rhododendron, gold peony, Guilv No.1, gold bud and Bixiangzao extracted from 72 parts of tea plant germplasm resources in the step (3) as templates, and detecting PCR amplification products by utilizing polyacrylamide gel vertical electrophoresis and a rapid silver staining method.

And (3) PCR reaction system: the total reaction volume is 20 mu L, wherein 2 XPCR Mix is 10 mu L, tea tree DNA template (1-50 ng) is 1 mu L, ddH₂O8. mu.L, 1. mu.L each of the forward primer (10. mu. mol/L) and the reverse primer (10. mu. mol/L).

PCR reaction procedure: pre-denaturation at 94 ℃ for 5min, followed by denaturation at 94 ℃ for 45s at high temperature in each cycle, annealing at 55-60 ℃ for 45s, extension at 72 ℃ for 7min after 35 cycles, and permanent storage at 4 ℃.

Electrophoretic color development: 5 μ L of PCR product was electrophoresed on 8% native polyacrylamide gel at constant voltage 200v for 60 min. After electrophoresis, the gel was stained with silver nitrate and developed. Washing the gel with ddH2O for 3 times, wherein each time is 1min, and then adding 0.5-1.5% of silver nitrate to dye for 10-15 min; and then washed 3 times with ddH2O for 1min each time. The gel is shaken gently in the developing solution until the developing band is clear, and then ddH is added₂And O, terminating the development. The electrophoresis results of the primer screening are shown in FIG. 1.

The PCR amplification electrophoresis staining result is shown in figure 1, and only the fragments amplified by the two pairs of SSR primers, namely 4CL-14 and 4CL-15, in the 6 pairs of SSR primers are different, the polymorphism is good, and the main band is clear.

TABLE 24 SSR primers for Coumaroyl CoA ligase (4CL) genes

(5) And (3) performing PCR amplification reaction by using 4CL-14 and 4CL-15 as SSR primers and 72 parts of tea plant genome DNA extracted in the step (4) as a template to research genetic polymorphism of tea plant germplasm resources, and obtaining polymorphism map characteristics as shown in the figure 2 and the figure 3 (the number of strips is 11 and 16).

Sequence listing

<110> Guangxi Zhuang nationality autonomous region subtropical zone crop research institute (Guangxi subtropical agricultural product processing research 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