A molecular marker primer for the identification of Actinidia eriantha variety Ganmi No. 6, and a method for its development and application

Abstract

The present invention discloses a molecular marker primer for the variety identification of Actinidia eriantha 'Ganmi No. 6'. The molecular marker is named EST-Ad42. The EST-Ad42 molecular marker includes a molecular marker primer EST-Ad42-F and a molecular marker primer EST-Ad42-R. The structure of the molecular marker primer EST-Ad42-F is gttaatttgatcgggatgg, and the structure of the molecular marker primer EST-Ad42-R is gaggagcttgagctgctat. The annealing temperature is 62°C. Another problem to be solved by the present invention is to provide a development method and an application method for the molecular marker primer for the variety identification of Actinidia eriantha 'Ganmi No. 6'. The molecular marker primer of the present invention has high specificity. The DNA is amplified using the molecular marker EST-Ad42 primer pair to construct a specific DNA molecular identification card.

Description

Technical Field

[0001] The present invention belongs to the technical fields of molecular biology and genetic breeding, and particularly relates to molecular marker primers for the variety identification of Actinidia eriantha 'Ganmi No. 6'. The present invention also relates to the development and application methods of molecular marker primers for the variety identification of Actinidia eriantha 'Ganmi No. 6'. Background Art

[0002] Kiwi fruit is a characteristic berry tree of the genus Actinidia in the family Actinidiaceae native to China. The fruit has a high nutritional density, diverse flower colors and is highly ornamental. It is a perennial deciduous vine fruit tree that combines fresh food and ornamental value. At present, there are 54 species and 21 varieties of Actinidia plants in the world. The fresh fruit of kiwi fruit is rich in ascorbic acid and amino acids. At present, it is mainly for fresh food in the market, and can also be processed into preserved fruits, fruit wines, fruit juices, etc., all of which have unique flavors and are therefore deeply favored by the public. Actinidia eriantha is an important species in the genus Actinidia unique to China, widely distributed in Zhejiang, Fujian, Jiangxi, Hunan, Guangxi, Guangdong, Guizhou and other places. It is a characteristic berry with great development potential after Actinidia chinensis and Actinidia deliciosa, and the wild germplasm resources are very rich. Due to factors such as the narrow distribution range, diploid nature, and gene conservation of Actinidia eriantha, Actinidia eriantha shows a relatively high degree of consistency in appearance. It is difficult to distinguish by traditional methods such as observing tree vigor, leaf morphology, phenological period, fruit shape, and flower organ color. The frequent introduction of kiwi fruit in the planting area has made the kiwi fruit varieties more mixed, and there are generally phenomena of different plants with the same name and the same plant with different names.

[0003] Using molecular marker technology to identify varieties has become the most efficient, accurate and environmentally insensitive method at present. Molecular marker technology has been studied in some citrus, pomegranate, grape, and peach, and can be used for genetic relationship analysis, germplasm identification, etc. Among them, SSR molecular marker technology is the most widely used. The construction of molecular identity cards using SSR has been successful in many species. The construction of molecular identity cards can not only identify germplasms with close genetic relationships, but also be a very important means for the protection of intellectual property rights.

[0004] 'Ganmi No. 6' is a variety selected from the natural variation population of wild Actinidia eriantha. The fruit is long cylindrical, and the fruit surface is densely covered with white short hairs. The fruit is medium-sized, with an average single fruit weight of 72.5 g. The pulp is dark green, the soluble solid content is 13.6%, the titratable acid content is 0.87%, the dry matter content is 17.3%, the ascorbic acid content is 7.23 mg / g, and the fruit maturity period is late October. When the fruit ripens to an edible state, it is easy to peel, the flesh is tender and fragrant, and the flavor is moderately sour and sweet. By developing molecular markers suitable for Actinidia eriantha 'Ganmi No. 6', it is beneficial to identify varieties at the molecular level and is more conducive to the protection of intellectual property rights. Summary of the Invention

[0005] The object of the present invention is to provide a molecular marker primer for the variety identification of Actinidia eriantha 'Ganmi No. 6' by using molecular marker technology. Another object of the present invention is to provide a method for the development and application of the molecular marker primer in the variety identification of kiwifruit.

[0006] To achieve the above object, the present invention takes the following technical measures:

[0007] A molecular marker primer for the variety identification of Actinidia eriantha 'Ganmi No. 6', characterized in that the molecular marker is named EST-Ad42, and the EST-Ad42 molecular marker includes two primers, namely molecular marker primer EST-Ad42-F and molecular marker primer EST-Ad42-R. The structure of the molecular marker primer EST-Ad42-F is gttaatttgatcgggatgg, the structure of the molecular marker primer EST-Ad42-R is gaggagcttgagctgctat, and the annealing temperature is 62 °C.

[0008] Another problem to be solved by the present invention is to provide a method for the development of a molecular marker primer for the variety identification of Actinidia eriantha 'Ganmi No. 6', and the steps include

[0009] (1) Download all kiwifruit EST sequences from the NCBI db EST database, and use SSR Hunter 1.3 software to screen the downloaded EST sequences for SSR;

[0010] (2) The criteria for SSR screening are: dinucleotide, trinucleotide, tetranucleotide, pentanucleotide and hexanucleotide, and the minimum number of repeats are 7 times, 5 times, 4 times, 4 times and 3 times respectively to obtain the intermediate sequence;

[0011] (3) Use Primer 5.0 software to design kiwifruit EST-SSR primers;

[0012] (4) Use the designed and synthesized primers to perform PCR amplification on Actinidia eriantha 'Ganmi No. 6' variety, and screen to obtain the molecular marker EST-Ad42. The EST-Ad42 molecular marker includes two primers, namely molecular marker primer EST-Ad42-F and molecular marker primer EST-Ad42-R. The structure of the molecular marker primer EST-Ad42-F is gttaatttgatcgggatgg, the structure of the molecular marker primer EST-Ad42-R is gaggagcttgagctgctat, and the annealing temperature is 62 °C.

[0013] To achieve better technical effects, in step (3), the method for designing kiwifruit EST-SSR primers is to select the intermediate sequence of 200 - 300 bp obtained in step (2), input it into Primer 5.0 software for design, and the design parameters are: primer sequence 22 ± 2 bp, product size 150 - 200 bp, primer GC content 40 - 55%, annealing temperature 50 - 65 °C.

[0014] Another problem to be solved by the present invention is to provide an application method of molecular marker primers for the variety identification of Actinidia eriantha 'Ganmi No. 6', and its steps include,

[0015] Use the above molecular marker primers EST-Ad42-F and molecular marker primers EST-Ad42-R to amplify the DNA of 'Ganmi No. 6' and reported varieties such as 'Huate', 'Ganlv No. 1', and 'Ganlv No. 2', detect it using 8 (w / v)% acrylamide gel, and develop the image by silver nitrate staining. Each band (DNA fragment) in the gel pattern is used as a molecular marker, and each polymorphic band can be regarded as an allele. Calculate the binary data of all loci according to the migration rate and presence or absence of each molecular marker. Record the presence of a band as '1' and the absence of a band as '0'. Draw a 1 / 0 matrix based on the size and number of different bands, construct a specific molecular identity card, and identify 'Ganmi No. 6' through the specific molecular identity card.

[0016] To achieve better technical effects, distinguish 'Huate', 'Ganlv No. 1', and 'Ganlv No. 2' through the specific molecular identity card.

[0017] Compared with the prior art, the advantages of the present invention are:

[0018] 1. Morphological markers are simple and intuitive, but are easily affected by environmental conditions, and some traits are difficult to define. Molecular markers are easy to operate, plant tissues can be obtained at any time for detection; there are many of them, covering the entire genome; they have high polymorphism, and there are many allelic variations among varieties.

[0019] 2. The primers of the present invention can amplify DNA fragments of different sizes in kiwifruit series varieties. This molecular marker amplification is stable and can be used for the variety identification of Actinidia eriantha 'Ganmi No. 6'.

[0020] 3. The molecular marker primers provided by the present invention have high specificity and a wide range of applications. They can identify the variety of Actinidia eriantha 'Ganmi No. 6' from 30 different varieties of kiwifruit. Use the molecular marker EST-Ad42 primers to amplify DNA and construct a specific DNA molecular identity card.

[0021] 4. Distinguish 'Huate', 'Ganlv No. 1' and 'Ganlv No. 2' kiwifruit according to the size and number of different bands.

[0022] Molecular marker primer EST-Ad42-F structure: gttaatttgatcgggatgg;

[0023] Molecular marker primer EST-Ad42-R structure: gaggagcttgagctgctat. Specific implementation mode

[0024] For the technical solutions described in the present invention, unless otherwise specified, they are all conventional solutions in the art; for the reagents or materials, unless otherwise specified, they are all sourced from commercial or public channels.

[0025] Example 1

[0026] A molecular marker primer for the variety identification of Actinidia eriantha 'Ganmi No. 6', characterized in that the molecular marker is named EST-Ad42, the EST-Ad42 molecular marker includes two primers, namely molecular marker primer EST-Ad42-F and molecular marker primer EST-Ad42-R, the structure of the molecular marker primer EST-Ad42-F is gttaatttgatcgggatgg, the structure of the molecular marker primer EST-Ad42-R is gaggagcttgagctgctat, and the annealing temperature is 62 °C.

[0027] Example 2

[0028] A method for developing a molecular marker primer for the variety identification of Actinidia eriantha 'Ganmi No. 6', the steps of which include,

[0029] (1) Download all Actinidia EST sequences from the NCBI db EST database, and use SSR Hunter 1.3 software to screen the downloaded EST sequences for SSRs;

[0030] (2) The criteria for SSR screening are: dinucleotides, trinucleotides, tetranucleotides, pentanucleotides and hexanucleotides, with the minimum number of repeats being 7 times, 5 times, 4 times, 4 times, and 3 times respectively, to obtain intermediate sequences;

[0031] (3) Use Primer 5.0 software to design Actinidia EST-SSR primers;

[0032] The design method of Actinidia EST-SSR primers is to select the intermediate sequences of 200 - 300 bp obtained in step (2), input them into Primer 5.0 software for design, and the design parameters are: primer sequence 22 ± 2 bp, product size 150 - 200 bp, primer GC content 40 - 55%, annealing temperature 50 - 65 °C;

[0033] (4)Using the designed and synthesized primers, perform PCR amplification on the Actinidia eriantha 'Ganmi No. 6' variety, and screen to obtain the molecular marker EST-Ad42. The EST-Ad42 molecular marker includes two primers, namely the molecular marker primer EST-Ad42-F and the molecular marker primer EST-Ad42-R. The structure of the molecular marker primer EST-Ad42-F is gttaatttgatcgggatgg, and the structure of the molecular marker primer EST-Ad42-R is gaggagcttgagctgctat. The annealing temperature is 62 °C.

[0034] Example 3

[0035] An application method of molecular marker primers for the identification of Actinidia eriantha 'Ganmi No. 6' variety, characterized in that

[0036] Use the above molecular marker primer EST-Ad42-F and molecular marker primer EST-Ad42-R to amplify the DNA of 'Ganmi No. 6' and reported varieties such as 'Huate', 'Ganlv No. 1', and 'Ganlv No. 2'. Detect using 8 (w / v)% acrylamide gel, and develop with silver nitrate staining. Each band (DNA fragment) in the gel pattern is used as a molecular marker, and each polymorphic band can be regarded as an allele. Calculate the binary data of all loci according to the migration rate and presence or absence of each molecular marker. A band is recorded as '1', and no band is recorded as '0'. Draw a 1 / 0 matrix according to the size and number of different bands, construct a specific molecular identification card, and identify 'Ganmi No. 6' through the specific molecular identification card.

[0037] In order to obtain better technical effects, distinguish 'Huate', 'Ganlv No. 1', and 'Ganlv No. 2' through the specific molecular identification card.

[0038] Example 4

[0039] An application method of molecular marker primers for the identification of Actinidia eriantha 'Ganmi No. 6' variety, for the application in kiwifruit breeding, the steps include

[0040] (1)Extract the DNA samples of 30 different varieties of kiwifruit respectively according to the CTAB method and dilute them to 150 ng / μl;

[0041] (2)Referring to the following PCR reaction system, add the corresponding reagents to a sterilized 200 μl centrifuge tube, mix well with a vortex mixer, and then centrifuge at 2000 rpm for 1 min;

[0042] ;

[0043] In the table, the upstream primer is the molecular marker primer EST-Ad42-F: gttaatttgatcgggatgg, and the downstream primer is the molecular marker primer EST-Ad42-R: gaggagcttgagctgctat;

[0044] (3) Refer to the following PCR reaction program and use a PCR instrument for amplification;

[0045] ;

[0046] (4) Use a pipette to inject the PCR product into the prepared 8 (w / v)% acrylamide gel for detection, silver nitrate staining and development, scan the image and save it to the computer, and estimate the molecular weight of the amplification product with 50 bp DNA ladder as a reference;

[0047] (5) Read the band information between 200 - 300 bp molecular weight. Each band (DNA fragment) in the gel map is used as a molecular marker, and each polymorphic band can be regarded as an allele. Calculate the binary data of all loci according to the migration rate and presence or absence of each molecular marker. A band is recorded as "1" and no band is recorded as "0";

[0048] (6) Use the molecular marker EST-Ad42 to amplify kiwifruit varieties, draw a 1 / 0 matrix according to the size and number of different bands, construct a specific molecular identification card, as shown in Table 1, effectively identify Actinidia eriantha 'Ganmi 6' and distinguish 'Huata';

[0049] .

[0050] Sequence Listing

[0051] <110> Jiangxi Agricultural University

[0052] <120> A Molecular Marker Primer for Identifying Actinidia eriantha 'Ganmi 6' Variety and Its Development and Application Method

[0053] <160> 2

[0054] <170> SIPOSequenceListing 1.0

[0055] <210> 1

[0056] <211> 19

[0057] <212> DNA

[0058] <213> Actinidia eriantha

[0059] <400> 1

[0060] gttaatttga tcgggatgg 19

[0061] <210> 2

[0062] <211> 19

[0063] <212> DNA

[0064] <213> Actinidia eriantha

[0065] <400> 2

[0066] gaggagcttg agctgctat 19 Sequence Listing <110> Jiangxi Agricultural University <120> Molecular Marker Primer for Variety Identification of Actinidia eriantha 'Ganmi 6' and Its Development and Application Method <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 19 <212> DNA <213> Actinidia eriantha <400> 1 gttaatttga tcgggatgg 19 <210> 2 <211> 19 <212> DNA <213> Actinidia eriantha <400> 2 gaggagcttg agctgctat 19

Claims

1. Application of a molecular marker primer in the identification of Actinidia eriantha cv. Ganmi No. 6 Characterized in that the molecular marker primer is EST-Ad42-F and EST-Ad42-R, the nucleotide sequence of EST-Ad42-F is gttaatttgatcgggatgg, the nucleotide sequence of EST-Ad42-R is gaggagcttgagctgctat, and EST-Ad42-R and EST-Ad42-F are used to distinguish Actinidia eriantha cv. Ganmi No. 6, Actinidia chinensis cv. Huate, Actinidia chinensis cv. Ganlv No. 1 and Actinidia chinensis cv. Ganlv No. 2