CN107354150B

CN107354150B - Differential sequence for identifying tomato fruit dry juice character, molecular marker and identification method thereof

Info

Publication number: CN107354150B
Application number: CN201710524590.1A
Authority: CN
Inventors: 刘磊; 李君明; 舒金帅; 朱庆宝; 邓学斌; 冯晶晶; 白金瑞
Original assignee: Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences
Current assignee: Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences
Priority date: 2017-06-30
Filing date: 2017-06-30
Publication date: 2020-04-17
Anticipated expiration: 2037-06-30
Also published as: CN107354150A

Abstract

The invention discloses a difference sequence of tomato fruit dry juice characters, which is shown as SEQ ID NO: 1, and also provides a method and a molecular marker for identifying the traits, which comprise the nucleotide sequence SEQ ID NO: 2, such as the nucleotide sequence of SEQ ID NO: 3 and provides a method for identifying the characters: (1) using the genomic DNA of the tomato to be identified as a template, and carrying out PCR amplification by using the molecular marker; (2) and (3) carrying out agarose gel electrophoresis detection on the amplification product, and if the PCR amplification product of the tomato to be identified contains the nucleotide sequence shown as SEQ ID NO: 1, the tomato to be identified can be identified as a tomato with juice, otherwise, the tomato is a tomato with dry juice. The molecular marker-assisted selection of the dry tomato fruit juice character is carried out by using the method, so that the selection efficiency can be improved, and the breeding process can be accelerated; the invention can complete the identification of the sample only by simple DNA extraction, PCR specific amplification and agarose gel electrophoresis detection.

Description

Differential sequence for identifying tomato fruit dry juice character, molecular marker and identification method thereof

Technical Field

The invention belongs to the technical field of genetics and biology, and particularly relates to a difference gene sequence of tomato fruit dry juice traits, a staged marker primer and an identification method thereof.

Background

The tomato has higher nutritional value and economic value, is an important fresh food product and processing raw material, has become the seventh crop in the world after corn, rice, wheat, potato and the like, and has an industrial production value of 500 billion dollars in the world year. Tomato fruit contains not only carbohydrates, cellulose, mineral elements, etc. which are beneficial to human body, but also various vitamins, and various sugars, organic acids and volatile organic compounds which determine the flavor of the fruit. Tomatoes are a model plant for studying fleshy fruits, particularly fleshy berries, and their development because of their important agricultural value, abundant nutritional value, superior phytological properties, and basic research advantages. Explains the physiological, developmental and metabolic mechanisms of many fruits, such as the molecular basis for organogenesis, development and maturation of fleshy fruits, as well as sensory quality and nutrition.

By utilizing continuously developed theories and technologies, excellent varieties meeting the production and market requirements are cultivated, and the method is the fundamental purpose of tomato breeding. With the development of genetics, genetic markers are widely applied and popularized, and are classified into phenotypic markers such as morphological markers, cytological markers, biochemical markers and the like, and genotypic markers such as DNA fragment markers and the like. The DNA fragment marker can directly reflect certain difference of genome between biological individuals or populations, is not influenced by environment and gene expression, and has the advantages of abundant quantity, stable heredity, simple operation and the like. From the late stage of the 80 s in the 20 th century, molecular marker assisted selection technology and genetic engineering technology as core biological breeding technology have been rapidly developed. The commonly used molecular markers include Restriction Fragment Length Polymorphism (RFLP) markers based on Southern hybridization, random amplified polymorphic DNA markers (RAPD) based on a DNA amplification method of PCR technology, specific sequence amplification markers (SCAR) and simple repeat sequence markers (SSR), amplified fragment length polymorphism markers (AFLP) based on PCR combined with enzyme digestion, cleaved amplified polymorphic sequence markers (CAPS), Single Nucleotide Polymorphisms (SNP), and the like. PCR is the English abbreviation for Polymerase Chain Reaction (Polymerase Chain Reaction), and PCR can amplify a DNA fragment. PCR technology has been widely used in biology, molecular biology, biochemistry, biomedicine, and other disciplines.

After ripening in common tomatoes, the fruit ventricular tissue develops into a gelatinous ventricular jelly (loculargel). The ventricular jelly is a typical characteristic of the fleshy berries such as tomatoes and plays an important role in the formation, development, flavor composition, texture change and the like of the fruits. The ventricular jelly, which is the second most abundant tissue in tomato fruit except the pericarp, is an important component of flavor quality. The dry tomato juice material (alf) fruit does not form ventricular jelly, the fruit can still normally develop and mature, the dry matter content of the dry tomato juice fruit is high, the storage and transportation resistance of the fruit can be improved, the degradation speed of tomato slices can be slowed down, and the dry tomato juice material (alf) fruit is used for making dry tomatoes, hamburger sandwiches and the like. The research of the dry juice tomato material can provide a theoretical basis for deeply understanding the formation and genetic improvement of the flavor quality of the tomato and lay a foundation for analyzing the molecular mechanism of the formation and development of the ventricular jelly of the tomato fruit. However, in the prior art, it is reported that molecular techniques are adopted to directly identify whether the tomato is a dry juice tomato.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a differential sequence of tomato fruit dry juice characters, a molecular marker and an identification method thereof, and aims to provide a method and a detection technology for rapidly screening and identifying the tomato fruit dry juice characters in a tomato seedling stage, which can rapidly and stably screen and identify the tomato fruit dry juice characters in an early plant stage.

In order to achieve the purpose, the technical scheme disclosed by the invention is as follows: the invention provides a difference sequence of tomato fruit dry juice characters, which is shown as SEQ ID NO: 1 is shown.

The invention also discloses a molecular marker for identifying the dry tomato fruit juice character, which comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 2, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 3.

the method for identifying the dry tomato fruit juice character by adopting the differential sequence and the molecular marker comprises the following steps: (1) using the genomic DNA of the tomato to be identified as a template, and carrying out PCR amplification by using the molecular marker of the gene sequence to obtain an amplification product;

(2) and (3) carrying out agarose gel electrophoresis detection on the amplification product, if the PCR amplification product of the tomato to be identified contains SEQ ID NO: 1, the tomato to be identified can be identified as a tomato with juice, otherwise, the tomato without juice.

The PCR reaction temperature program in the step (1) is as follows: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min, and 30 cycles; extension at 72 ℃ for 8 min.

And (3) carrying out agarose gel electrophoresis detection on the amplification product in the step (2), wherein an electrophoresis display band only displays a 358bp band, and the tomato to be identified is a dry juice tomato.

And (3) carrying out agarose gel electrophoresis detection on the amplified product in the step (2), wherein the electrophoresis displays a 497bp band or two 497bp and 358bp bands, and the tomato to be identified is a juicy tomato.

The amplification kit in the step (1) comprises:

the invention has the beneficial effects that: the dry juice tomato plant is obtained by carrying out auxiliary selection on a tomato breeding population by adopting the primer set, which shows that the molecular marker is practical and effective for molecular marker auxiliary selection of the dry tomato juice character, and the molecular marker auxiliary selection carried out by the invention can improve the selection efficiency and accelerate the breeding process.

The method is used for identifying whether the tomato fruits have the dry juice character, and the identification of the samples can be completed only by simple DNA extraction, PCR specific amplification and agarose gel electrophoresis detection. The method does not need restriction enzyme digestion, and has the advantages of economy, simple operation, strong specificity, good repeatability and the like.

Drawings

FIG. 1 shows the electrophoresis result of PCR product of molecular primer pair for identifying dry juice of tomato fruit of the present invention,

m is 100bp DNA ladder, p1 is female parent H1706, p2 is male parent 06790, F₁F of H1706X 06-790₁The rest is F of H1706 multiplied by 06-790₂And (6) numbering strains.

Detailed Description

The present invention is described in further detail below with reference to specific examples.

The first embodiment is as follows: the invention provides a difference sequence of tomato fruit dry juice characters, which is shown as SEQ ID NO: 1, the molecular marker for identifying the character comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 2, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 3, respectively.

The invention obtains a batch of SNP loci and insertion deletion fragments linked with the dry juice character by re-sequencing the dry juice tomato genome, and carries out genetic linkage analysis and verification by designing primers for the loci and the fragments, thereby screening a series of molecular markers linked with the dry juice character, including CAPS, dCAPS, PCR markers and the like. As the dry-juice tomato is recessive, co-dominant markers including CAPS-1, CAPS-2 and dCAPS-1, etc. were selected for validation.

CAPS-1：

A forward primer: 5'-TAGGCAAACTCTATTCATCAAGG-3' (CAPS1 No.1)

Reverse primer: 5'-CTATGCTGTGGTAGGTAGACTTGC-3' (CAPS1 No.2)

After PCR amplification, restriction with restriction enzyme MboI followed by agarose electrophoresis, the dried tomato juice could not be cleaved, showing a 480bp band. The common tomato juice can be subjected to enzyme digestion or partial enzyme digestion, and shows two bands of 120bp and 360bp, or shows three bands of 120bp, 360bp and 480 bp.

CAPS-2：

A forward primer: 5'-TGAGAAAAATAAGAATAGCCGATG-3' (CAPS2 No.1)

Reverse primer: 5'-CACACATCAACCTCCAGACTCC-3' (CAPS2 No.2)

After PCR amplification, restriction enzyme TaqI is used for enzyme digestion, agarose electrophoresis is carried out, the dried tomato juice can be completely enzyme digested, and two bands of 290bp and 340bp are displayed. The common tomato can not be cut by enzyme or can not be completely cut by enzyme, and shows a 630bp band, or shows three bands of 290bp, 340bp and 630 bp.

dCAPS-1：

A forward primer: 5'-TTATGAAAACATTCGGTGGTGC-3' (dCAPS1 No.1)

Reverse primer: 5'-ATAATTTGATGTTTAATTTAGCCAT-3' (dCAPS1 No.2)

After PCR amplification, the tomato is cut by restriction enzyme BccI, polyacrylamide gel electrophoresis is needed, the tomato in dry juice cannot be cut by enzyme, and only one 180bp strip is displayed. The common tomato juice can be cut by enzyme, and one band of 160bp is shown, or two bands of 160bp and 180bp are shown.

dCAPS-2：

A forward primer: 5'-GAGATGTACATCATATACTTACATG-3' (dCAPS2 No.1)

Reverse primer: 5'-TTGTTCGTGTATTCAAACGCTTAT-3' (dCAPS2 No.2)

After PCR amplification, the tomato is cut by restriction enzyme AflIII, polyacrylamide gel electrophoresis is needed, the tomato can be completely cut by enzyme, and only one 150bp band is displayed. The common tomato juice can not be cut by enzyme or can not be completely cut by enzyme, and one band of 170bp or two bands of 150bp and 170bp are shown.

Because the restriction enzyme digestion needs to be carried out after PCR of the primers, such as CAPS, dCAPS and the like, the time consumption is long, the price of the restriction enzyme is high, and the digestion time, the temperature and the like can cause incomplete digestion, the accuracy of an electrophoresis strip is influenced, and the accuracy of dry juice character selection and identification is influenced.

Therefore, the invention selects the primer marker which does not need enzyme digestion for verification, and the difference sequence and the molecular primer of the dried tomato fruit juice character disclosed by the invention are nucleotide sequences which are most accurate and simple and convenient in method for identifying the dried tomato fruit juice character.

Example two: the invention discloses a method for identifying the dry tomato juice character, which comprises the following steps: (1) using the genomic DNA of the tomato to be identified as a template, and carrying out PCR amplification by using the molecular marker of the gene sequence to obtain an amplification product;

The PCR reaction temperature program in the step (1) is as follows: pre-denaturation at 4 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 1min, and 30 cycles; extension at 72 ℃ for 8 min.

And (3) carrying out agarose gel electrophoresis detection on the amplification product in the step (2), wherein the electrophoresis display band only displays a 446bp band, so that the tomato to be identified is a dry juice tomato.

And (3) carrying out agarose gel electrophoresis detection on the amplification product in the step (2), wherein an electrophoresis display band displays a 862bp band or displays two bands of 862bp and 446bp, and the tomato to be identified is a juicy tomato.

The amplification kit in the step (1) comprises:

the mass concentration and the molar concentration of each component of the kit are the mass concentration and the molar concentration of each component dissolved in the corresponding solvent, and each component is dissolved in the corresponding solvent to form a solution for detection.

The following tests show that the differential sequence, the designed primer and the identification method provided by the invention are the most accurate, effective and convenient for identifying the dry tomato juice character, and H1706 multiplied by 06790F₂The strain is obtained according to the following method: h1706 (as the female parent) and 06790 (as the male parent) to give F₁，F₁Selfing harvest F₁Obtaining F from the seeds of the plant₂The seed of (1). Get F at random₂Planting the seeds of (1) to obtain F₂The plant is numbered as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 2,28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45。

And (I) carrying out normal cultivation management in the field, identifying the dry juice character of each strain of tomato after the color of the tomato is changed, and investigating 10 fruits by each strain to ensure the accuracy of the identification result. The field identification results are shown in table 1, indicating that

line numbers

4, 15, 16, 20, 24, 25, 28, 29, 31, 32, 40 and 42 are dry juice tomatoes; the

strain numbers

1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 17, 18, 19, 21, 22, 23, 26, 27, 30, 33, 34, 37, 38, 39, 41, 43, 44 and 45 are common juicy tomatoes.

Watch 1

Line number

PCR amplification product

Fruit character

Line number

PCR amplification product

Fruit character

1

862bp

With juice

25

446bp

Dry juice

2

862bp

With juice

26

862bp

With juice

3

862bp

With juice

27

862bp

With juice

4

446bp

Dry juice

28

446bp

Dry juice

5

862bp+446bp

With juice

29

446bp

Dry juice

6

862bp

With juice

30

862bp+446bp

With juice

7

862bp+446bp

With juice

31

446bp

Dry juice

8

862bp+446bp

With juice

32

446bp

Dry juice

9

862bp+446bp

With juice

33

862bp

With juice

10

862bp+446bp

With juice

34

862bp

With juice

11

862bp+446bp

With juice

35

862bp+446bp

With juice

12

862bp+446bp

With juice

36

862bp+446bp

With juice

13

862bp+446bp

With juice

37

862bp+446bp

With juice

14

862bp

With juice

38

862bp

With juice

15

446bp

Dry juice

39

862bp+446bp

With juice

16

446bp

Dry juice

40

446bp

Dry juice

17

862bp+446bp

With juice

41

862bp+446bp

With juice

18

862bp

With juice

42

446bp

Dry juice

19

862bp+446bp

With juice

43

862bp+446bp

With juice

20

446bp

Dry juice

44

862bp+446bp

With juice

21

862bp

With juice

45

862bp

With juice

22

862bp+446bp

With juice

F₁

862bp+446bp

With juice

23

862bp+446bp

With juice

p1

862bp

With juice

24

446bp

Dry juice

p2

446bp

Dry juice

(II) identifying the characters of the dry juice of the fruits in the adult plant period in the field

After the PCR reaction was completed, the PCR products of the primer pair were subjected to 1.5% agarose gel electrophoresis detection, Goldview staining, buffer 0.5 XTBE, electrophoresis at 4V. cm-1 constant pressure for 1h, and observation and photography by Bio-Rad gel imaging system. The preparation method of 10 × TBE (1L) is as follows: 108g of Tris, 55g of boric acid and 7.44g of EDTA are metered to 1L by ddH2O, and a 0.5 Xworking solution is prepared by distilled water before use and is stored at normal temperature. The electrophoresis results of the PCR products of the primer pairs are shown in FIG. 1, and specific bands were obtained from the PCR products of H1706 (strain No. p1), 06790 (strain No. p2) and strains No. 1-45.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

SEQUENCE LISTING

<110> vegetable and flower institute of Chinese academy of agricultural sciences

<120> differential sequence of tomato juice dry juice characters, molecular marker and identification method thereof

<130>

<160>1

<170>Patentln version 3.3

<210>1

<211>416

<212> differential sequences of tomato fruit Dry juice traits

<213>

<220>

<223>

<400>1

TATTTTTAAT TTAAAAGCTA TTTTTTTAAG CCAATCCAGA CGGTCTCTTA ATATACAGGT 60

CAAACCTCAT TAAATAAAAT TTAAATATTT GAAAGAAAAG TTTGAGAGAT TTTAAACAGC 120

ACAAGGGGCA TATTAGTCAA GAAGAAACAA AAATAACACG CTTTGCAATA ATTGGTGAAA 180

TTTTAGTCTG CAATAAACAA TCCCATAACA TCACGTCTGG TTTATATCTG GAAAAAAGCC 240

ATTTGAATGT CATTTTCTTG GCCAGCCATC TCTATTATCT CTCTTCACTT TAATTTTGAG 300

TGATACTTTC TTCGTCCATC CGACTCAACA CACATCTTTT AAGAAATAAT AAATTCGAAG 360

AGTAATTTTA TTATATATCA TCAGTCACCC CTATTGGTAA CACGTCATCT AAATAT 416

<210>2

<211>

<212> upstream primer for identifying tomato fruit dry juice character

<213>

<220>

<223>

<400>2

5’-CCGTTTGAATAGGTTTAGTAGTCG-3’

<210>3

<211>

<212> downstream primer for identifying tomato fruit dry juice character

<213>

<220>

<223>

<400>3

5’-CTGAAAACTCACACAACTTCCAAA-3’

Claims

1. A differential sequence of tomato fruit dry juice character is characterized in that the sequence is shown as SEQ ID NO: 1 is shown.

2. The primer for identifying the dry tomato fruit juice character is characterized by comprising an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 2, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 3, respectively.

3. A method for identifying the characteristics of dried tomato fruit juice is characterized by comprising the following steps:

(1) carrying out PCR amplification by using an amplification kit containing genomic DNA of a tomato to be identified as a template and a primer of a nucleotide sequence shown in claim 2 to obtain an amplification product;

(2) detecting the amplification product, if the PCR amplification product of the tomato to be identified contains the PCR amplification product of the tomato to be identified as shown in the SEQ ID NO: 1, the tomato to be identified can be identified as a common tomato with juice, otherwise, the tomato with dry juice is identified.

4. The method for identifying the dry juice character of the tomato fruits as claimed in claim 3, wherein the PCR reaction temperature program in the step (1) is: pre-denaturation at 4 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 1min, and 30 cycles; extension at 72 ℃ for 8 min.

5. The method for identifying the dry juice character of the tomato fruits as claimed in claim 3, wherein the amplification product is detected by agarose gel electrophoresis in the step (2), and the tomato to be identified is a dry juice tomato if the electrophoresis band shows only a 446bp band.

6. The method for identifying the dry tomato fruit juice character in the claim 3, wherein the agarose gel electrophoresis detection is carried out on the amplification product in the step (2), the electrophoresis band shows a 862bp band or shows two bands of 862bp and 446bp, and the tomato to be identified is a tomato with juice.

7. The method for identifying the dry juice character of the tomato fruits as claimed in claim 3, wherein the amplification kit in the step (1) comprises:

the mass concentration of the genomic DNA template of the tomato to be identified is 25 ng/mu l, the mass concentration of the upstream primer is 50 ng/mu l, the mass concentration of the downstream primer is 50 ng/mu l, the concentration molar mass concentration of a dNTPs mixture is 10mM, and MgC1 is added₂The molar mass concentration of the solution is 25mM, the mass concentration of Taq DNA polymerase is 5U/μ l, and the mass concentration and the molar concentration of each component forming the kit are the mass concentration and the molar concentration of each component dissolved in the corresponding solvent.