Method for establishing SSR (Simple Sequence Repeat) fingerprint spectrum of broad beans

A technology of fingerprints and construction methods, which is applied in the field of identifying broad bean varieties by fingerprints of different varieties of broad beans, and the construction of SSR fingerprints of broad beans. Achieve the effects of preventing counterfeit and inferior varieties from entering the market, protecting crop varieties, and easy operation

Active Publication Date: 2017-05-31
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is simple and economical, but because many morphological traits have a long identification period and are easily affected by the environment, it cannot accurately and quickly provide identification basis for new variety infringement cases, and there are often deviations in the understanding of certain traits by different testers , leading to the

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  • Method for establishing SSR (Simple Sequence Repeat) fingerprint spectrum of broad beans
  • Method for establishing SSR (Simple Sequence Repeat) fingerprint spectrum of broad beans
  • Method for establishing SSR (Simple Sequence Repeat) fingerprint spectrum of broad beans

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 is used to identify the screening of SSR and EST-EST primers of broad bean varieties

[0050] In this example, the core genome SSR and EST-SSR primers were synthesized by analyzing the genome sequence and EST sequence of broad bean by using the genomic DNA of 48 varieties of broad bean (see Table 1) popularized in the main producing areas of broad bean in my country as templates. After screening, the molecular marker combinations composed of 15 pairs of genomic SSR and 6 pairs of EST-SSR primers can completely identify the differences of 48 broad bean varieties.

[0051] In the final screening, 15 pairs of genomic SSR molecular markers and 6 pairs of EST-SSR molecular markers were obtained.

[0052] Table 2 21 pairs of SSR core primers

[0053]

[0054]

[0055] The primers in the above Table 1 respectively correspond to SEQ ID NO.1-42 in the sequence listing.

Embodiment 2

[0056] Example 2 Construction of characteristic fingerprints of broad bean varieties and establishment of identification methods for broad bean varieties

[0057] 1. Total DNA extraction of broad bean varieties

[0058] The improved CTAB method was used to extract the DNA of the tested variety: take about 2g of fresh young leaves, grind them into powder in liquid nitrogen, put them into a 2.0ml centrifuge tube, and store them in a -80°C refrigerator for later use; preheat to 65°C 2×CTAB extract at ℃, add β-mercaptoethanol at a ratio of 1% of the solution volume, and mix thoroughly; take out the ground leaf tissue, add 800 μL of preheated CTAB extract to each sample, and vortex 1-2min, 65°C water bath for 1h (turn it upside down once every 15min, mix well); after the water bath heating, add 800μL chloroform / isoamyl alcohol (24:1) solution to the centrifuge tube, mix for 15-20min, Then centrifuge at 10,000 rpm for 15 minutes; absorb about 600 μL of supernatant and transfer it t...

Embodiment 3

[0107] Embodiment 3 Application of the present invention's identification method for broad bean varieties

[0108] Extract the leaf DNA of 6 broad bean samples with known variety names, and carry out PCR amplification of the DNA of the broad bean varieties to be checked with 21 pairs of primers in the molecular marker combination screened in Example 1 of the present invention; with reference to Example 2 of the present invention PCR reaction system, PCR reaction program, polyacrylamide gel electrophoresis and variation site recording method (method 1), and repeated experiments at the same time using fluorescence capillary electrophoresis and variation site recording method (method 2), the fingerprint code obtained and The characteristic fingerprints (Table 5-Table 16) of broad bean varieties obtained in the present invention were compared to verify the accuracy of the identification method established in Example 2 of the present invention.

[0109] As a result, it was found th...

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Abstract

The invention provides a method for establishing an SSR (Simple Sequence Repeat) fingerprint spectrum of broad beans, and belongs to the technical field of plant variety identification and breeding. The method provided by the invention provides a marker combination consisting of 21 pairs of broad bean genome SSR primers, and the nucleotide sequences of which are shown in SEQ ID NO. 1 to 42 respectively; the broad bean varieties are identified by PCR (Polymerase Chain Reaction) amplification technology, non-denaturing polyacrylamide gel electrophoresis or capillary electrophoresis detection technology, and the identification of broad bean seed varieties and genetic diversity assessment work can be completed in a short time. The method has the advantages of time saving, high efficiency, fastness, accuracy, and convenience in operation, and the identification results are not easily affected by the environment. The method provided by the invention has the advantages of effectively monitoring the authenticity of the broad bean seeds, revealing the genetic variation and genetic relationship of the varieties from the DNA level, protecting the crop varieties, preventing the fake and inferior varieties from entering the market, and also providing a technical support for the reasonable utilization of excellent germplasm during the breeding of broad bean varieties, and has good application prospects.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and plant variety identification, in particular to a method for constructing broad bean SSR fingerprints and a method and application for identifying broad bean varieties using the constructed fingerprints of different varieties of broad beans. Background technique [0002] Broad bean (Vicia faba L.), as a grain, vegetable, feed, and green manure crop, occupies an important position in my country's crops. Broad beans are produced and distributed in all continents of the world, and more than 43 countries around the world produce broad beans. Broad beans have been cultivated in my country for more than 2,000 years. Except for Shandong, Hainan and the three northeast provinces, which are rarely planted, broad beans are planted in other parts of the country. In the past 10 years, the planting area of ​​broad beans in China has reached more than 1.4 million hectares 2 , accounting for abou...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6895C12Q2600/156C12Q2531/113C12Q2565/125
Inventor 宗绪晓张红岩杨涛刘荣
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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