Application of EST-SSR marker primer combination and screening method in germplasm genetic diversity analysis of dwarf type and sprawl type of peas

A technology of genetic diversity and screening method, which is applied in the application field of genetic diversity analysis of pea dwarf and trailing germplasm, can solve the problems of pea EST information scarce, restricting the development of EST-SSR molecular markers, and slow development.

Inactive Publication Date: 2015-11-11
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, pea EST information is scarce and develops slowly (as of March 2015, there were only 21,838 pea ESTs in the NCBI database), which sever

Method used

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  • Application of EST-SSR marker primer combination and screening method in germplasm genetic diversity analysis of dwarf type and sprawl type of peas
  • Application of EST-SSR marker primer combination and screening method in germplasm genetic diversity analysis of dwarf type and sprawl type of peas
  • Application of EST-SSR marker primer combination and screening method in germplasm genetic diversity analysis of dwarf type and sprawl type of peas

Examples

Experimental program
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Effect test

Embodiment 1

[0041] The application of EST-SSR marker primer combination and screening method on pea dwarf and vine germplasm genetic diversity analysis, its specific steps include as follows:

[0042] (1) Preparation of germplasm materials.

[0043] The pea materials utilized in the present invention include 8 parts of dwarf peas and 12 parts of vine pea materials, as shown in Table 1 below.

[0044] Table 1 Pea germplasm information table.

[0045]

[0046] (2) High-throughput transcriptome sequencing

[0047] Using dwarf and vine peas as materials, total RNA was extracted from the plant, and mRNA was isolated and purified using Oligotex® mRNAkit (Qiagen); cDNA was synthesized by reverse transcription using mRNA as a template; Fragments were constructed to construct a high-quality cDNA library; the cDNA samples were sequenced using a next-generation high-throughput sequencing platform (454GSFLX).

[0048] (3) DNA extraction of germplasm samples

[0049] Seedlings of the tested g...

Embodiment 2

[0059] In conjunction with Example 1, the result analysis of the application of EST-SSR marker primer combination and screening method on pea dwarf and vine germplasm genetic diversity analysis is as follows:

[0060] figure 1 For the detection results of EST-SSR labeled fluorescent capillary electrophoresis, through the analysis of 170 pairs of primers, such as figure 1 As shown, a total of 46 pairs of polymorphic primers were obtained, and obvious bands could be obtained in the same lane, see figure 2 .

[0061] As shown in Table 2 below, 12 pairs of ETS-SSR markers were screened and used for identification of vegetable pea germplasm resources.

[0062] like image 3 As shown, 8 dwarf-type and 12 vine-type vegetable peas were successfully divided into two categories. And to identify their kinship, see Figure 4 .

[0063]

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Abstract

The invention discloses an EST-SSR marker primer combination, which includes 12 pairs of EST-SSR available primers. A screening method of the EST-SSR marker primer combination comprises the steps of SSR locus screening in an EST sequence obtained through sequencing of a transcriptome of pea, primer design, PCR amplification, capillary electrophoresis detection to a product and marker screening. The EST-SSR marker primer combination is applied in germplasm genetic diversity analysis of dwarf type and sprawl type of peas. The EST-SSR marker primer combination can be used for quickly and accurately distinguishing different germplasm materials of peas and analyzing the genetic relationship, thereby providing a basis of oriented seed breeding of the varieties.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to the application of an EST-SSR marker primer combination and a screening method thereof in the analysis of pea dwarf and vine germplasm genetic diversity. Background technique [0002] Pea (Pisum. sativum L.) is a diploid (2n=2x=14) leguminous plant and is the second largest edible legume crop in the world. Vegetable peas, also known as sweet peas, are deeply loved by consumers because of their sweet and delicious seeds, unique flavor and rich nutrition. The share in my country's agricultural product processing and export market is expanding day by day, and the market prospect is broad. However, the existing vegetable peas in my country are mainly imported varieties with serious aging, and there is a lack of high-quality and high-yielding varieties independently bred. As a typical representative of vines, the main varieties of vegetable peas in the Chinese market are ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 徐盛春龚亚明胡齐赞刘娜
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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