SSR primer set and method for seed purity identification of hot pepper variety F1 hybrid-Huifeng No. 2

A pepper and variety technology, applied in the directions of biochemical equipment and methods, recombinant DNA technology, microbial determination/inspection, etc., can solve the problems of application limitation, large environmental impact, low accuracy, etc., and achieve the effect of high commercial application value

Active Publication Date: 2016-03-02
INST OF VEGETABLES GUANGDONG PROV ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the purity identification of pepper seeds is usually planted in field plots and observed by morphology. However, due to the long growth period of peppers, morphological identification usually takes 60-120 days. The cycle is long, greatly affected by the environment, and the accuracy is low. financial resources, so its application is limited

Method used

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  • SSR primer set and method for seed purity identification of hot pepper variety F1 hybrid-Huifeng No. 2
  • SSR primer set and method for seed purity identification of hot pepper variety F1 hybrid-Huifeng No. 2
  • SSR primer set and method for seed purity identification of hot pepper variety F1 hybrid-Huifeng No. 2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] From April to July 2015, 96 plants of 'Huifeng No. 2' hybrid first generation planted in Guangzhou Zhongluotan Experimental Base were morphologically identified. At the same time, each individual plant was numbered and sampled, and the purity was identified in the laboratory using the present invention. The specific operation is as follows:

[0063] (1) Genomic DNA of 96 pepper seedlings was extracted by TPS method:

[0064] ①Put about 200mg of pepper seedling leaves into 2.0ml centrifuge tubes, insert a plastic grinding rod in each tube, clamp it with long-handled tweezers in liquid nitrogen for about 30 seconds, quickly grind it into powder, and then add 800μl TPS to extract liquid;

[0065] ②Water bath at 72°C for 30 minutes;

[0066] ③Centrifuge at 12000rpm for 10min-15min at room temperature;

[0067] ④Take the supernatant, transfer it to a 1.5ml centrifuge tube, add an equal volume of isopropanol, and centrifuge at 12000rpm for 5min;

[0068] ⑤ Discard the sup...

Embodiment 2

[0074] In March 2015, Guangdong Kenong Vegetable Seed Industry Co., Ltd. entrusted our laboratory to conduct seed purity identification before selling the 'Huifeng No. 2' hybrid. Randomly selected 300 seeds to grow seedlings in seedling cups, numbered and sampled about 10 days after emergence , 292 strains were sampled altogether, carried out DNA extraction, PCR amplification, electrophoresis detection to 292 single strains to be checked by the method of example 1, the result analysis showed that altogether 288 single strains had detected the characteristic band of length 141bp and 149bp simultaneously, and These 288 individual plants also detected the characteristic bands of 196bp and 208bp at the same time, indicating that these 288 individual plants were true hybrids, and the purity was: 288 / 292*100%≈98.6%. Excluding the seedling time, the entire identification process takes 1 person and 2 days to complete, and the testing cost for each sample is about 0.15 yuan.

Embodiment 3

[0076] Purchase the seeds of its other main pepper varieties 'Yuehong 1', 'Huifeng 1' and 'Huifeng 5' from Guangdong Kenong Vegetable Seed Industry Co., Ltd., grow seedlings in seedling cups, and extract plant DNA from individual plants in about 10 days According to the method and steps of Example 1, 2 pairs of primer sets were used to simultaneously amplify 'Yuehong No. 1', 'Huifeng No. 1', 'Huifeng No. 2' and 'Huifeng No. 5' 4 pepper varieties, and the results showed that 2 When the primer set is amplified simultaneously, each variety has its own specific band combination, which can be distinguished from the other 3 varieties (see image 3 ), therefore, the two pairs of primers can not only identify the seed purity of the pepper variety 'Huifeng No. 2', but also identify the authenticity of the above four pepper varieties at the same time.

[0077] The above examples show that the method of the present invention can accurately and reliably distinguish the hybrid of pepper va...

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Abstract

The invention discloses an SSR primer set and method for seed purity identification of the hot pepper variety F1 hybrid-Huifeng No. 2. The SSR primer set comprises SSR molecular markers L0143 and L0622 and primer pairs corresponding to the SSR molecular markers. Any primer pair can be utilized for distinguishing the hot pepper variety F1 hybrid-Huifeng No. 2 from the female parent and the male parent of the hot pepper variety F1 hybrid-Huifeng No. 2, and can rapidly detect the seed purity of the hot pepper variety F1 hybrid-Huifeng No. 2. In addition, the primer set can also be used for seed authenticity identification of hot pepper varieties F1 hybrid-Yuehong No. 1, Capsicum annuum L.syn.C.frutescens L and F1 hybrid-Huifeng No. 5. The method has the advantages of being rapid, accurate, low in cost, easy to implement and the like, can replace a traditional hybrid seed purity identification method, and has the high commercial application value.

Description

technical field [0001] The invention relates to a molecular marker technology, in particular to a primer set and a method for detecting the seed purity of pepper variety 'Huifeng No. 2' by using SSR molecular markers. Background technique [0002] 'Huifeng 2' is a hybrid pepper variety of the first generation. It is medium and early maturing, has the characteristics of storage and transportation, and good quality. It is also resistant to epidemic diseases and has strong heat, cold, waterlogging and drought tolerance. Wang Hengming, Li Ying, Guo Hanquan, et al. A new pepper variety 'Huifeng No. 2' with early to medium maturity and high quality [J]. Acta Horticultural Science, 2010, 02: 337-338.). Since pepper is a common cross-pollinated crop, it is mainly self-crossed, and there are some natural hybrids (<50%) at the same time. Therefore in F 1 Even if other methods of seed production (such as the use of male sterility) are used for seed production, artificial assisted ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 徐晓美王恒明程雪丽李颖孙保娟谢大森
Owner INST OF VEGETABLES GUANGDONG PROV ACAD OF AGRI SCI
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