Method and special primer set for identifying facticity to-be-tested variety belonging to CCRI 63

A primer pair and auxiliary identification technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve problems such as counterfeiting, affecting the promotion effect of hybrid cotton, and affecting seed sales

Active Publication Date: 2014-08-27
INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the hybrid seed production of cotton is still dominated by artificial seed production, and the sterile line seed production has not been promoted. With the continuous rise of labor costs, the cost of hybrid seed production is getting higher and higher, and the quality of seeds is getting worse. The hybrid cotton seeds sold are seriously faked. Some F2 replaces F1, some conventional cotton replaces F1, and there are female parent doping, etc., so that the hybrids cannot achieve the desired effect of increasing production in the promotion, and affect the promotion effect of hybrid cotton.
Conventional field identification takes a long time, which will affect the sales of seeds in the current year, and the identification effect is not very satisfactory due to the impact of the environment during the Jiadai identification in Hainan

Method used

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  • Method and special primer set for identifying facticity to-be-tested variety belonging to CCRI 63
  • Method and special primer set for identifying facticity to-be-tested variety belonging to CCRI 63
  • Method and special primer set for identifying facticity to-be-tested variety belonging to CCRI 63

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1. Construction of the DNA fingerprint of China Cotton Institute 63

[0030] 1. Screening of SSR primer pairs

[0031] After a large number of cotton SSR primer screenings, it was found that 30 SSR primer pairs (nucleotide sequences are shown in Table 1) can be used to make the DNA fingerprint of China Cotton Institute 63.

[0032] Table 130 nucleotide sequences of primer pairs

[0033] Serial number

Primer name

Forward primer (5’→3’)

Reverse primer (5’→3’)

1

CIR62

TTTAGAGGAGAAGTTTAGG (sequence 1)

CAGTCTCTTGTAGTTTCATT (sequence 2)

2

CS62

GATGGCTACCTCCCTTTGTA (sequence 3)

CGTAAGGAAGCCTAGCAAAA (sequence 4)

3

NAU1070

ACCAACAATGGTGACCTCTT (sequence 5)

CCCTCCATAACCAAAAGTTG (Sequence 6)

4

NAU1102

ATCTCTCTGTCTCCCCCTTC (serial 7)

GCATATCTGGCGGGTATAAT (Sequence 8)

5

NAU1186

AATGGTCCTGCTCCAGATT (sequence 9)

AATCGTCGTCGTCGAATTAT (sequence 10)

6

NAU2026

GAATCTCGAAAACCCCATCT (sequence 11)

ATTTGGAAGCGAAGTACCAG (sequence 12)

7

NAU2173

GCCAAATAGGTCACACA...

Embodiment 2

[0073] Example 2. Application of 30 SSR primer pairs to assist in the identification of China Cotton Institute 63

[0074] The following cotton varieties were tested: Zhongzhimian No. 2 (Institute of Plant Protection, Chinese Academy of Agricultural Sciences), Ruiza 816 (Jinan Xinrui Seed Industry), Handan 8266 (Handan Academy of Agricultural Sciences), China Cotton Institute 63 ( China Cotton Seed Industry).

[0075] Detection method: extracting genomic DNA of cotton leaves, using genomic DNA as a template, respectively using the 30 SSR primer pairs for PCR amplification, and then performing 8% polyacrylamide gel electrophoresis and silver staining for color development.

[0076] The PCR system and PCR reaction procedures are the same as in Example 1.

[0077] See electrophoresis Figure 31 (1, 2, 3, 4 represent Zhongmian Institute 63, Zhongzhimian 2, Ruiza 816 and Han 8266, respectively). See Table 3 for the banding results.

[0078] Table 3 Band patterns after amplification of geno...

Embodiment 3

[0113] Example 3. Using 20 primer pairs among 30 SSR primer pairs to identify seed purity

[0114] A batch of China Cotton Institute 63 is collected from the seed production company (in the form of seeds, a total of 200 seeds; the seeds are theoretically China Cotton Institute 63, but usually due to the limited operation level in the seed production process, it is difficult to achieve 100% purity. Female parent plant phenomenon or other adulteration phenomenon), randomly select 26 seeds from 200 seeds, extract the genomic DNA of the seeds, use the genomic DNA as the template, and use 20 SSR primer pairs (see the table for specific SSR primer pairs) 4) Perform PCR amplification, and then perform 8% polyacrylamide gel electrophoresis and silver staining for color development. Using the genomic DNA of the male parent of China Cotton Institute 63 (2 replicate samples) as a template, respectively, 20 SSR primer pairs were used for PCR amplification, and then 8% polyacrylamide gel elec...

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Abstract

The invention discloses a method and a special primer set for identifying facticity of to-be-tested variety belonging to CCRI 63. The method provided by the invention comprises the following steps: respectively carrying out PCR (Polymerase Chain Reaction) amplification by use of CIR62, CS62, NAU1070, NAU1102, NAU1186, NAU2026, NAU2173, NAU2277, NAU2343, NAU1196, NAU1085, NAU1255, NAU1369, NAU1233, NAU1269, NAU2272, NAU1187, NAU1375, CS51, NAU1028, NAU2342, NAU868, HAU1300, NAU6094, DPL0238, NAU1362, NAU859, HAU2786, CGR6410 and NAU0478 primers, if a PCR amplification product meets more than 28 standards in the standards of 1)-30), and the to-be-tested cotton is CCRI 63, wherein the genomic DNA of to-be-tested cotton is taken as a template. The method disclosed by the invention can be applied to controlling the quality of cotton seeds, ensuring the quality of commercially available CCRI 63 seeds and fighting against counterfeiting CCRI 63 seeds.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a method for identifying whether the variety to be tested is China Cotton Institute 63 and its special primer set. Background technique [0002] Cotton has obvious heterosis. Hybrid cotton varieties have been used for many years, and the effect of increasing yield is obvious. It has been popularized and applied in a large area. At present, hybrid seed production of cotton is still dominated by artificial seed production, and sterile line seed production has not been promoted. With the continuous increase in labor costs, the cost of hybrid seed production is getting higher and higher, and the quality of seeds is getting worse and worse. The sales of hybrid cotton seeds are seriously fraudulent. Some F2 replaces F1, some conventional cotton replaces F1, and there are phenomena such as female parent adulteration, which makes the hybrids not reach the expected yield increase effect...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q2531/113C12Q2565/125
Inventor 付小琼叶武威杨付新黄殿成
Owner INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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