A kasp molecular marker, kit and application for identifying male sterility restoration gene of pepper cms

A molecular marker, gene recovery technology, applied in recombinant DNA technology, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of high cost, distinguish polymorphism, time-consuming, etc., to save manpower and accuracy high effect

Active Publication Date: 2022-07-19
河南省农业科学院园艺研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since SNP is a polymorphism caused by single nucleotide variation, conventional PCR technology and gel electrophoresis technology cannot be used to distinguish its polymorphism
However, using sequencing technology to analyze SNP sites, although the marker density is high, the cost is relatively high and it is time-consuming

Method used

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  • A kasp molecular marker, kit and application for identifying male sterility restoration gene of pepper cms
  • A kasp molecular marker, kit and application for identifying male sterility restoration gene of pepper cms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Taking Chaotian pepper numbered PC324 as an example, 15 individual plants (among which, 5 were sterile, 3 were homozygous and fertile, and 7 were heterozygous and fertile) were selected from the F2 generation genetic segregation population as the experimental material for KASP markers. reliability test.

[0078] 1. Extract the total genomic DNA of the tested peppers by 2X CTAB method, measure the DNA concentration of the sample with Nanodrop, and adjust the final concentration of the sample DNA to 80-100ng / μL.

[0079] 2. Then construct a 5 μL PCR system: 2.5 μL of the sample DNA obtained in step 1, 0.07 μL of mixed primers with a concentration of 50 μM and 2.5 μL of 2x Mastermix; the mixed primers include the following three primers mixed in equal proportions:

[0080] Fluorescently labeled upstream primer F1 (SEQ ID NO. 4):

[0081] GAAGGTGACCAAGTTCATGCT AAGATCTGTGAGCCCAACGA

[0082] Fluorescently labeled upstream primer F2 (SEQ ID NO. 5):

[0083] GAAGGTCGGAGTCA...

Embodiment 2

[0089] Using the SNPline genotyping platform of LGC Company, 384 different types of pepper materials were selected as experimental materials to test the reliability of KASP markers.

[0090] 1. Extract the total genomic DNA of the tested peppers by 2X CTAB method, measure the DNA concentration of the sample with Nanodrop, and adjust the final concentration of the sample DNA to 80-100ng / μL;

[0091] 2. Construct a 5μL PCR system on the SNPline of LGC Company: it contains 2.5μL of the DNA to be tested obtained in step 1, 0.14μL of mixed primers with a concentration of 50μM and 5μL of 2x Master mix; the mixed primers include the following three primers mixed in equal proportions:

[0092] Fluorescently labeled upstream primer F1 (SEQ ID NO. 4):

[0093] GAAGGTGACCAAGTTCATGCT AAGATCTGTGAGCCCAACGA

[0094] Fluorescently labeled upstream primer F2 (SEQ ID NO. 5):

[0095] GAAGGTCGGAGTCAACGGATT AAGATCTGTGAGCCCAACGT

[0096] Downstream primer R (SEQ ID NO. 3):

[0097] SEQ ID ...

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Abstract

The present invention provides a KASP molecular marker for identifying CMS male sterility restoration gene in pepper, a kit and application thereof, and relates to the technical field of molecular marker-assisted breeding. The KASP molecular marker comprises upstream primer F1, upstream primer F2 and downstream primer R The primer set was amplified. Using the KASP molecular marker of the present invention can quickly complete the identification of the fertility of the pepper CMS isolated population, quickly determine whether the pepper contains the CMS fertility restoration gene, can carry out batch experiments, save a lot of manpower, and is especially suitable for the operation of a large number of samples . The invention provides a kit for identifying the CMS restoration gene of pepper and the identification method with high accuracy.

Description

technical field [0001] The invention relates to the technical field of molecular marker-assisted breeding, in particular to a KASP molecular marker for identifying the CMS male sterility restoration gene in pepper, a kit and its application. Background technique [0002] The use of CMS (Cytoplasmic male sterility) three-line matching seed production in pepper can save artificial emasculation, improve the purity of seeds, and be beneficial to the protection of intellectual property rights. However, if the fertility recovery of the male parent is not strong, it will seriously affect the fruit shape and yield of F1 (Wang Lihao, 2007). Therefore, selection and breeding of restorer lines (male parent) with strong recovery, high combining ability and excellent agronomic traits has become one of the main problems to be solved urgently in pepper CMS heterosis utilization (Shen Huolin and Shi Zhengqiang, 2002). The traditional selection of pepper CMS fertility genes is based on the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/6858C12N15/11
Inventor 张强姚秋菊张涛常晓轲韩娅楠程志芳刘卫王彬任福森郭志伟孙强陈昊放刘贺娟焦禹顺
Owner 河南省农业科学院园艺研究所
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