CN109825637B

CN109825637B - Primer for identifying purity of Saijing 316 seeds of non-heading Chinese cabbage and application

Info

Publication number: CN109825637B
Application number: CN201910280785.5A
Authority: CN
Inventors: 黄志银; 王超楠; 李梅; 华德平; 张红; 范伟强; 张斌; 刘晓晖
Original assignee: Tianjin Kerun Agricultural Science & Technology Co ltd
Current assignee: Tianjin Kerun Agricultural Science & Technology Co ltd
Priority date: 2019-04-09
Filing date: 2019-04-09
Publication date: 2022-07-05
Anticipated expiration: 2039-04-09
Also published as: CN109825637A

Abstract

The invention discloses an InDel molecular marker specific primer for detecting purity of Jun 316 non-heading Chinese cabbage hybrid seeds, which is a nucleotide sequence shown as SEQ ID NO. 1-2. The invention further discloses a method for identifying the purity of the handsome 316 non-heading Chinese cabbage hybrid seed by using the specific primer. The specific primer and the method provided by the invention can complete the purity identification of the Saijian 316 hybrid seeds of the Saijian cabbage within 3-5 hours in a laboratory, have the advantages of rapidness, stability, no environmental influence and the like, can replace the traditional field seed purity identification method, and are beneficial to the efficient and accurate quality control of the Saijian 316 Saijian cabbage hybrid seeds.

Description

Primer for identifying purity of Saijing 316 seeds of non-heading Chinese cabbage and application

Technical Field

The invention belongs to the technical field of biology, and relates to a primer sequence for identifying purity of a Sujun 316 hybrid seed of a non-heading Chinese cabbage and application thereof.

Background

Chinese cabbage without heading (Brassica rapa L. ssp. chinensisMakino) belongs to brassica crops of cruciferae, also called pakchoi, green vegetables and rapes (northern areas of China), which are vegetables planted and supplied all the year round originally in the middle and lower reaches of Yangtze river and in the southern areas of China, become important nationwide green-leaf vegetables, and the planting area in Japan, Korea and other foreign countries is also increasing.

The traditional seed purity identification method for the non-heading Chinese cabbages generally adopts field planting, and the purity of the seeds is identified by observing the growth characteristics when the seeds grow to a specific period, so that the method has the defects of long period, large labor amount, easiness in environmental influence and the like. With the development of molecular biology, the application of molecular marker assisted breeding technology in agricultural breeding is wider and wider, and the InDel molecular marker can be used for directly identifying the difference of genome DNA levels among samples and identifying the purity of seeds, and has the advantages of short time consumption, good repeatability, no environmental influence and the like.

Sujun 316 is one of the series of non-heading Chinese cabbage hybrids developed by vegetable research institute of Tianjin scientific and technical GmbH, and has the characteristics of high growth speed, early girdling, beautiful plant shape, high yield, long suitable harvesting period, good quality and the like. The variety has bright green leaf color, short leaf stalk, wide and bright leaf, no wax powder, high disease resistance and wide adaptability. The seed purity of the variety is always identified by adopting a traditional field method, and the conflict between the seed purity identification time and the sale period is often caused. A rapid and accurate seed purity identification method needs to be developed to adapt to the popularization of Jun 316 and the increase of the seed consumption.

Disclosure of Invention

The invention aims to provide an InDel marked primer sequence for detecting seed purity of Jun 316 non-heading Chinese cabbage hybrid and application thereof, and the invention adopts the following technical scheme to realize the purpose:

a specific primer for detecting the purity of Jun 316 Neisseria cepharantha hybrid seeds is characterized by being a nucleotide sequence shown in SEQ ID NO. 1-2:

ATG CTT GGA GAT GAG ACA TGG ATC AAG SEQ ID NO:1

TTG CTC ATA TCA CCA TCA TAC AGA TAG GG SEQ ID NO:2。

the invention further discloses a method for detecting the purity of the Jun 316 non-heading Chinese cabbage hybrid seeds by adopting the specific primers, which is characterized by comprising the following steps of:

(1) extracting genome DNA of a sample to be detected of the Sujun 316 hybrid;

(2) carrying out PCR amplification by taking the genomic DNA of the sample as a template and the labeled primer as a specific primer; the reaction system is as follows: a10 μ L system comprising: 10 × PCR Buffer, 1.0 μ L; 2.5 mM dNTP, 0.2 μ L; 10 μmol L-1 primers, 0.4 μ L; 5U, mu L-1 Taq, 0.2 mu L; 50 ng, muL-1 DNA, 1.5 muL; ddH2O, 6.7 μ L. The amplification procedure used was: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 s, renaturation at 57 ℃ for 30 s, extension at 72 ℃ for 30 s, and 35 cycles; final extension at 72 deg.C for 5 min; the specific primers are as follows:

ATG CTT GGA GAT GAG ACA TGG ATC AAG SEQ ID NO:1

TTG CTC ATA TCA CCA TCA TAC AGA TAG GG SEQ ID NO:2；

(3) performing polyacrylamide gel electrophoresis separation and silver staining on a product obtained by the PCR amplification;

(4) counting the silver staining results: the seeds containing the male parent and female parent double-parent material strips are hybrid seeds, the seeds containing only the female parent or male parent material strips are non-hybrid seeds, and the detected purity of the fast Jun 316 hybrid (%) = (the total grain number of the detected seeds-the number of the non-hybrid seeds)/(the total grain number of the detected seeds) × 100%.

The invention further discloses an application of the detection method in the aspect of quickly and accurately detecting the purity of the Jun 316 non-heading Chinese cabbage hybrid seeds. The experimental results show that: the method can quickly and accurately identify the purity of the handstand 316 non-heading Chinese cabbage hybrid seed.

The invention mainly solves the defects of long time consumption, easy environmental influence and the like of the traditional field identification method for the purity of the Sujun 316 non-heading Chinese cabbage hybrid seeds, and the main difficulty lies in developing a molecular marker primer which has polymorphism between a male parent and a female parent and has good stability and easy distinction between the male parent and the female parent of the Sujun 316 non-heading Chinese cabbage hybrid seeds.

Compared with the prior art, the specific primer for detecting the purity of the Jun 316 Chinese cabbage hybrid seed and the application thereof have the positive effects that:

through the specific primer and the application provided by the invention, the seed purity identification of the Saijin 316 hybrid can be completed within 3-5 hours in a laboratory, the method has the advantages of rapidness, stability, no environmental influence and the like, can replace the traditional field seed purity identification method, and is beneficial to the efficient and accurate quality control of the Saijin 316 commercial seeds and the further popularization of the variety.

Drawings

FIG. 1 is an electrophoretogram of InDel primer A10-2 in Jinshakuai No. 1 male parent, female parent and hybrid samples;

m represents DNA Marker, 1-3 are three repeated materials of a Saiji 316 male parent, 4-5 are three repeated materials of a Saiji 316 female parent, and 7-9 are three repeated materials of a Saiji 316 hybrid;

FIG. 2 is an electrophoretogram of 46 samples of Jun 316 hybrid species detected by InDel primer;

m represents DNA Marker, P₁Denotes the paternal material, P₂The female parent material is shown, the rest is hybrid material, wherein the position of the triangle shows that the material is non-hybrid material, and the rest is hybrid material.

Detailed Description

Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention. The experimental methods used in the following examples are all conventional methods unless otherwise specified; the instruments, materials, reagents, etc. used are commercially available unless otherwise specified. The used male parent material NS07 and female parent material KR08 can be claimed from the subject group of Chinese cabbage of vegetable research institute of Tianjin department agricultural science and technology, Inc., and the Sujun 316 non-heading Chinese cabbage hybrid is commercially available.

Example 1

First, screening of purity identification InDel primer

According to genome re-sequencing data of the Chinese cabbage, a plurality of pairs of primers are designed, polymorphism detection is carried out on three individual plants, namely Sujun 316, a male parent and a female parent, a primer pair A10-2 with codominant difference is screened out, and the sequence is as follows:

F:5’-ATG CTT GGA GAT GAG ACA TGG ATC AAG-3’(SEQ ID NO:1)

R:5’- TTG CTC ATA TCA CCA TCA TAC AGA TAG GG-3’(SEQ ID NO:2)

the mark has good repeatability and clear bands, PCR amplification is carried out by taking the primer as a specific primer and taking the genome DNA of a sample to be detected as a template, the amplification product is subjected to electrophoretic separation and dyeing detection, and the result shows that: a133 bp specific band can be amplified in a male parent material, a 104bp specific band can be amplified in a female parent material, and 133bp and 104bp specific bands can be simultaneously amplified in a Sujun 316 hybrid material, as shown in figure 1.

Purity identification of Sujun 316 seeds

1. Extraction of genomic DNA of sample to be tested

The materials are male parent and female parent materials, and are from Sujun 316 hybrid seeds produced by 5 users signed with a farmer in a seed production base. A CTAB method is utilized to extract leaf genome DNA of 92 plants of female parent material, male parent material and Sujun 316 hybrid to be detected, and the method comprises the following specific steps: taking 0.2 g of young leaves, putting the young leaves into a 1.5 mL centrifuge tube, adding 50 mu L of 2% CTAB extraction buffer solution, grinding, then filling the mixture to 400 mu L, and carrying out water bath at 65 ℃ for 30 min; add 400 μ L chloroform: isoamyl alcohol (24: 1), and shake gently for 5 min. Centrifuging at 12000 rpm for 5 min; collecting supernatant 200 μ L, adding precooled isopropanol 200 μ L, mixing, standing at-20 deg.C for 20 min; centrifuging at 12000 rpm for 10 min; discarding the supernatant, adding 150 μ L of precooled ethanol, gently mixing and cleaning, and centrifuging at 10,000 rpm for 5 min; discarding the supernatant, and air-drying or blow-drying; adding 100 mu L of distilled water to dissolve DNA, and standing for 1 h at room temperature; the DNA was diluted to 50 ng/. mu.L with distilled water and used as a PCR template or stored at-20 ℃ for future use.

PCR amplification: and carrying out PCR amplification by using the genome DNA of the sample to be detected as a template and the InDel primer provided by the invention as a specific primer to obtain an amplification product. The reaction system is as follows: a10 μ L system comprising: 10 × PCR Buffer, 1.0 μ L; 2.5 mM dNTP, 0.2 μ L; 10 μmol L-1 primers, 0.4 μ L; 5U, mu L-1 Taq, 0.2 mu L; 50 ng, muL-1 DNA, 1.5 muL; ddH2O, 6.7 μ L. The amplification procedure used was: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 s, renaturation at 57 ℃ for 30 s, extension at 72 ℃ for 30 s, and 35 cycles; final extension at 72 ℃ for 5 min.

3. Electrophoretic separation and staining of amplification results: and (3) carrying out electrophoretic separation on the PCR product by using 8% non-denaturing polyacrylamide gel electrophoresis, and carrying out silver staining for color development to count the size of an electrophoretic band.

4. And (4) analyzing and counting results: the electrophoresis strip of the PCR amplification product of the sample to be detected contains the strips of the male parent material amplification product and the female parent material amplification product at the same time, and is a Sujun 316 hybrid; the strip containing only the amplified product of the male parent or female parent material is the selfed seed of the male parent or female parent material, as shown in FIG. 2.

Through statistics, the seed purity of the handsome 316 hybrid from 5 users signed up by the breeding base is shown in table 1, wherein the purity of handsome 316 seed produced by farmer 1 is 94.6%, the purity of handsome 316 seed produced by farmer 2 is 98.9%, the purity of handsome 316 seed produced by farmer 3 is 100%, the purity of handsome 316 seed produced by farmer 4 is 97.8%, and the purity of handsome 316 seed produced by farmer 5 is 100%.

Compared with the traditional field identification result, the identification result of the purity of the Sujun 316 hybrid by using the InDel molecular marker provided by the invention is consistent with the field identification result, which shows that the seed purity of the Sujun 316 cabbage commercial variety can be quickly and accurately identified by the method.

TABLE 1 seed purity identification results of 5 farmers in Sujun 316 seed production base

Example 2

The traditional identification method comprises the following comparative tests: the method comprises the steps of randomly taking about 100 samples of Sujun 316 commercial varieties to be detected produced by 5 contracting farmers in a seed production base, ditching in open field, sowing according to the density of about 200 plants per square meter, discharging full seedlings about 3 days after sowing, judging whether the samples to be detected are Sunjiao fast No. 1 commercial varieties according to the fact whether the expression characters such as plant height, leaf color, leaf shape, leaf stalk color, length and width of the samples to be detected accord with the unity and consistency of the fast jun 316 commercial varieties or not when 7 leaves and 1 heart stage grow, and counting the seed purity of the samples to be detected.

And (4) conclusion: the result of identifying the purity of the seed of the Sagnan 316 Neisseria monocytogenes hybrid seed by the method is consistent with the result of identifying the seed purity by the traditional field, and meanwhile, the method has the advantages of quickness, no environmental influence and the like, so that the method can replace the traditional field identification method and is used for identifying the seed purity of the Sagnan 316 Neisseria monocytogenes commercial variety.

It will be understood by those skilled in the art that the foregoing specific embodiments are illustrative only and are not limiting upon the scope of the invention, and that all equivalent modifications and changes in light of the spirit of the invention will be suggested to persons skilled in the art and are to be included within the spirit and purview of the appended claims.

SEQUENCE LISTING

<110> Tianjin Kerun agriculture science & technology GmbH

<120> primer for identifying purity of Saijian 316 seeds of non-heading Chinese cabbage and application

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 27

<212> DNA

<213> Artificial sequence

<400> 1

atgcttggag atgagacatg gatcaag 27

<210> 2

<211> 29

<212> DNA

<213> Artificial sequence

<400> 2

ttgctcatat caccatcata cagataggg 29

Claims

1. A method for detecting purity of Jun 316 Chinese cabbage hybrid seeds by using a specific primer is characterized by comprising the following steps:

(1) extracting genome DNA of a sample to be detected of a Sujun 316 hybrid;

(2) carrying out PCR amplification by taking the genomic DNA of the sample as a template and an InDel labeled primer as a specific primer; the reaction system is as follows: a10 μ L system comprising: 10 × PCR Buffer, 1.0 μ L; 2.5 mM dNTP, 0.2 μ L; 10 μmol L-1 primers, 0.4 μ L; 5U. mu L-1 Taq, 0.2 mu L; 50 ng, muL-1 DNA, 1.5 muL; ddH2O, 6.7 μ L;

the amplification procedure used was: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 s, renaturation at 57 ℃ for 30 s, extension at 72 ℃ for 30 s, and 35 cycles; final extension at 72 deg.C for 5 min; the specific primers are as follows:

ATG CTT GGA GAT GAG ACA TGG ATC AAG SEQ ID NO:1

TTG CTC ATA TCA CCA TCA TAC AGA TAG GG SEQ ID NO:2；

(3) performing polyacrylamide gel electrophoresis separation and silver staining on the product obtained by PCR amplification, and simultaneously amplifying specific bands of 133bp and 104bp in a Sujun 316 hybrid material;

(4) counting the silver staining results: purity (%) of detected handsome 316 hybrid seed = (total grain number of detected seed-number of non-hybrid seed)/(total grain number of detected seed) × 100%.