Method for identifying vicia variety authenticity and special SSR primer combination thereof

A combination of primers and the technology of vetch, applied in the biological field, can solve the problems of reducing the efficiency and accuracy of detection, the inability to determine the molecular weight, and data comparison errors, etc., and achieve the effects of saving manpower, protecting rights and interests, and ensuring authenticity

Pending Publication Date: 2022-05-24
全国畜牧总站
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the silver staining method is greatly affected by the environment during the gel preparation process, and it is easy to cross samples during vertical electrophoresis, and the molecular weight of the separated fragments after development cannot be determined, which will cause errors in the data comparison of different batches of samples and reduce the detection efficiency. efficiency and accuracy

Method used

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  • Method for identifying vicia variety authenticity and special SSR primer combination thereof
  • Method for identifying vicia variety authenticity and special SSR primer combination thereof
  • Method for identifying vicia variety authenticity and special SSR primer combination thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0137] Example 1. Development of SSR primer combinations for identification of 9 national varieties of the genus Vetia using capillary electrophoresis fluorescence SSR fingerprints

[0138] 1. Plant the seeds of Jianghuai, culture at 25°C under alternating light and dark conditions, collect the leaves of 30 seedlings after 14 days, and mix samples of equal quality to obtain sample 1.

[0139] According to the above method, replace Jianghuai with Liangshan, Gongnong, Lanjian 2, Chuanbei, Lanjian 3, Qingshuihe, Yanbian and 333 / A, and the other steps are the same, and samples 2 to 9 are obtained in sequence.

[0140] 2. The genomic DNA of the samples (sample 1, sample 2, sample 3, sample 4 or sample 5) is extracted respectively by using the improved CTAB method to obtain the genomic DNA of the samples.

[0141] 3. The genomic DNA of the sample (sample 1, sample 2, sample 3, sample 4 or sample 5) was interrupted by ultrasound, and the DNA fragment of 400-600bp was recovered by cut...

Embodiment 2

[0151] Example 2. Using the SSR primer combination 1 developed in Example 1 to detect 9 nationally approved varieties of the genus Vetia

[0152] 1. Using the improved CTAB method to extract the genomic DNA of the samples (sample 1, sample 2, sample 3, sample 4 or sample 5) in Example 1 to obtain the genomic DNA of the sample.

[0153] 2. TP-M13-SSR PCR amplification

[0154] (1) Using the genomic DNA of the sample as a template, perform PCR amplification with the primer set VA42, primer set VA100, primer set VA107, primer set VA114 or primer set VA118 assembled in Example 1 to obtain PCR amplification products.

[0155]The reaction system is 10 μL, consisting of 1 μL sample genomic DNA (concentration 100-500 ng / μL), 0.5 μL forward primer (primer with “F” in the primer name) (concentration 10 μM), 0.5 μL reverse primer (primer Primers with “R” in their names) (concentration: 10 μM), 0.5 μL M13 universal primers (primers with “M13” in their names) (concentration: 10 μM), 1 μL ...

Embodiment 3

[0183] Example 3. Using the SSR primer combination 2 developed in Example 1 to detect 9 nationally approved varieties of the genus Vetia and the accuracy experiment

[0184] The seed to be tested a, the seed to be tested b, the seed to be tested c, the seed to be tested d, the seed to be tested e, the seed to be tested f, the seed to be tested g, the seed to be tested h and the seed to be tested i are phenotype identifications The commercially available seeds of Jianghuai, Liangshan, Gongnong, Lanjian 2, Chuanbei, Lanjian 3, Qingshuihe, Yanbian and 333 / A.

[0185] 1. Plant the seeds to be tested (the seeds to be tested a, the seeds to be tested b, the seeds to be tested c, the seeds to be tested d, the seeds to be tested e, the seeds to be tested f, the seeds to be tested g, the seeds to be tested h or the seeds to be tested i), 25 DEG C, alternate light and dark culture, after 14 days, collect leaves of 30 seedlings, and mix samples with equal mass to obtain samples to be tes...

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Abstract

The invention discloses a method for identifying the authenticity of vicia varieties and a special SSR (Simple Sequence Repeat) primer combination thereof. The SSR primer combination provided by the invention is composed of five primer pairs, each primer pair is composed of two primer sequences, and the nucleotide sequences of all primers in the five primer pairs are sequentially shown as SEQ ID NO: 1 to SEQ ID NO: 10. The SSR primer combination provided by the invention can be used for carrying out early identification on vicia varieties in a seed or seedling stage, so that the authenticity of the varieties is ensured, and the rights and interests of producers and breeders are practically protected. The method provided by the invention has the advantages of accuracy, low cost, simplicity in operation, manpower and material resource saving and the like, and has a very wide application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for identifying the authenticity of a variety of the genus Vetia and a combination of special SSR primers. Background technique [0002] The genus Vicia L. is an important group of plants in the Viciae family (Papilionatae) of the leguminosae (Leguminosae). Forage, green manure, traditional Chinese medicine and ornamental use value, among which the crude protein content of arrowhead pea and wild pea is higher than 20%, and the feeding value is comparable to that of alfalfa. However, the morphological similarities among species in the genus Vetia are relatively high and easy to be confused, and there are great controversies in the traditional morphological classification. And one of the main factors of development and utilization. At present, there are 9 varieties of the genus Vetto that have passed the national examination and approval, namely Jianghuai (purple...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/11C12Q1/686
CPCC12Q1/6895C12Q1/686C12Q2525/151C12Q2565/125C12Q2563/107Y02A50/30
Inventor 洪军高秋王显国刘芳林积圳吴菲菲李玉荣张义王梅娟
Owner 全国畜牧总站
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