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Gourd gourd core molecular marker set and its application developed based on kasp technology

A core molecule and marker set technology, which is applied in recombinant DNA technology, microbial determination/inspection, DNA/RNA fragments, etc. It can solve the problems of long working cycle, easy to be affected by human factors, etc., and achieve the effect of solving resource redundancy.

Active Publication Date: 2022-04-19
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the continuous advancement of the sorting and protection of gourd germplasm resources in my country and the increase in the number of gourd varieties bred year by year, a series of problems have emerged, including how to judge similar varieties, how to identify counterfeit varieties, how to judge the quality of seeds, and how to carry out quality research. Identification; the traditional identification and evaluation of gourd germplasm and varieties mainly rely on phenotype, which has a long working cycle and is easily affected by human factors, which has become one of the bottlenecks for the efficient use of germplasm resources

Method used

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  • Gourd gourd core molecular marker set and its application developed based on kasp technology
  • Gourd gourd core molecular marker set and its application developed based on kasp technology
  • Gourd gourd core molecular marker set and its application developed based on kasp technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] This example is the acquisition of gourd core molecular marker set.

[0063] Based on the sequenced and assembled "Hangzhou Long Melon" genome (version number is Lagenaria siceraria cv. 'HangzhouGourd'2.0) and GourdBase (http: / / www.gourdbase.cn / ), the first gourd-specific multi-omics database platform at home and abroad , evenly select SNP loci covering gourd chromosomes, 100 for each, 1100 in total. Resequencing was performed on 12 important gourd germplasms with large differences in geographical origin and phenotypic characteristics. Using the resequencing results of the important germplasm, 200 SNP sites with good polymorphism and uniform distribution on the chromosome were selected, and then primers were designed to convert the 200 SNP sites into KASP markers.

[0064] The 200 SNP markers screened out were used to detect the genotypes of 206 gourd germplasm materials (provided by the cowpea and gourd laboratory of Zhejiang Academy of Agricultural Sciences). Based o...

Embodiment 2

[0071] In this embodiment, the purity of the gourd hybrid 'Zhepu No. 9' to be tested is detected by using the gourd core SNP marker set.

[0072] The gourd hybrid 'Zhepu 9' is a new generation gourd hybrid bred by the cowpea and gourd research laboratory of the Institute of Vegetables, Zhejiang Academy of Agricultural Sciences, and it is also one of the main popularized varieties at present. The specific implementation steps are as follows:

[0073] 1) Extraction of DNA

[0074] 96 seeds of the gourd hybrid 'Zhepu No. 9' to be tested were randomly selected for planting and DNA was extracted. Specifically: take fresh leaves, cut them into pieces and put them into a 2mL centrifuge tube (1 / 3 is suitable), add a clean steel ball, freeze them in liquid nitrogen, and place them on a 2000-type GENO / GRINDER proofer until they are powdered (1100rpm vibration for 40s) ; Open the cap of the centrifuge tube, add 600 μL of DNA extraction solution (2xCTAB) to each corresponding tube, shak...

Embodiment 3

[0080] In this embodiment, the gourd core SNP marker set is used to detect whether the gourd variety to be tested is 'Zhepu No. 6'.

[0081] Based on the core SNP marker set of 22 gourds, the SNP fingerprint of gourd variety 'Zhepu 6' was constructed. Since the SNP markers are biallelic markers, the fingerprint composed of 22 SNP markers will have 44 bases , the core SNP fingerprint of gourd variety 'Zhepu 6' is: GAAATTCTCCCCCCTCTCCGACCTTAGAAACTTGGAATTTTCCGG. Use the barcode online generator (http: / / barcode.tec-it.com / barcode-generator.aspx) to convert the SNP fingerprint to the two-dimensional code to obtain the SNP fingerprint two-dimensional code of the gourd variety 'Zhepu No. 6', Such as image 3 shown.

[0082] The gourd varieties to be tested were randomly selected, and the DNA was prepared according to the steps in Example 2. At the same time, 22 core SNP marker sets were used for genotyping detection and their SNP fingerprints were obtained. The steps for performin...

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Abstract

The invention relates to the technical fields of molecular biology and plant molecular breeding, in particular to a gourd core molecular marker set developed based on KASP technology and its application; the gourd core molecular marker set contains 22 SNP markers for PCR amplification The primer pair of the gourd core molecular marker set contains 22 KASP primer sets, and each KASP primer set is used for PCR amplification of the corresponding SNP marker; Variety purity detection, gourd SNP fingerprint library construction, gourd genetic diversity analysis and gourd molecular marker-assisted breeding; the present invention develops a gourd core molecular marker set based on KASP technology, which can quickly, accurately and effectively identify Evaluating germplasm resource materials or bred varieties can solve problems such as resource redundancy, inconvenient preservation and management, and intellectual property disputes caused by the accumulation of a large number of germplasm.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and plant molecular breeding, in particular to a gourd core molecular marker set developed based on KASP technology and its application. Background technique [0002] In the prior art, with the development of DNA molecular marker technology, more and more crops begin to use different types of DNA molecular markers to identify the authenticity and purity of seeds. Among them, the most practical and prominent one is the new-generation DNA molecular marker technology SNP, which is widely distributed, high density, good stability, and suitable for large-scale screening. It has been applied to the identification of vegetable crops such as peppers, melons, watermelons, and cucumbers. Seed purity testing, etc. With the development of high-throughput genotyping technology, the Competitive allele-specific PCR (KASP) technology with higher SNP usage efficiency and analysis throughput came out, a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/6858C12N15/11
CPCC12Q1/6895C12Q1/6858C12Q2600/156C12Q2531/113C12Q2563/107
Inventor 汪颖吴晓花李艳伟吴新义汪宝根鲁忠富李国景
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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