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Kiwi fruit sex molecular marker primers based on fluorescent capillary electrophoresis and application thereof

A capillary electrophoresis and molecular labeling technology, applied in the field of molecular biology, can solve the problems of high technical threshold, high detection cost, low detection accuracy, poor practicability, etc., and achieve the effects of efficient identification, fast detection speed, and reduced detection cost.

Pending Publication Date: 2021-11-16
WUHAN ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

By searching the existing technologies at home and abroad, although there have been research reports on the early sex identification of kiwifruit from the aspect of molecular markers, the operation steps involve PAGE gel preparation, electrophoresis, silver staining and other processes, which are very cumbersome, technical threshold and detection cost Both are very high, the practicability is poor, and the existing detection accuracy is not high

Method used

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  • Kiwi fruit sex molecular marker primers based on fluorescent capillary electrophoresis and application thereof
  • Kiwi fruit sex molecular marker primers based on fluorescent capillary electrophoresis and application thereof
  • Kiwi fruit sex molecular marker primers based on fluorescent capillary electrophoresis and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Extraction of kiwifruit sex-specific sequences and development of marker primers

[0036] In this example, kiwifruit plants collected in the kiwifruit germplasm resource garden of Wuhan Academy of Agricultural Sciences were used as materials to develop sex-specific markers.

[0037] The applicant downloaded the whole genome data of Chinese kiwifruit 'Hongyang' through the CNBI database, and the floral organ transcriptome data of male and female samples, and determined the sex-determining segment by comparing differentially expressed sequences and gene homology mapping. As a result of gene structure annotation, genome and transcriptome fragments that differ between male and female are found. For the two specific sequences screened out, we designed PCR primers in the genomic regions of kiwifruit male samples, amplified the corresponding genomic sequences of different male samples, screened the conserved regions of the amplified fragments, and then developed a s...

Embodiment 2

[0052] Example 2 Comparison of the application effects of Ms1, Ms2 and existing molecular markers

[0053] (1) Source of primers

[0054] The molecules we developed were compared with existing molecular markers for early sex identification in Actinidia. A total of four labeled primers reported by Ms1 and Ms2 and Zhang Qiong et al. (Zhang Qiong et al., 2020, CN111394495A) and Qi Xiujuan et al. (Qi Xiujuan et al., 2019, CN109609686A) were selected for comparison. The corresponding primers were synthesized according to the primer sequences they provided.

[0055] (2) Comparison results of four markers

[0056] We used the PCR system given in the references, and the above two molecular markers (marked as marker1 by Qi Xiujuan et al., marked as marker2 by Zhang Qiong et al.) and the two pairs of molecular markers developed by us (Ms1, Ms2) in the same Under the conditions of electrophoresis detection and comparison, 96 female kiwifruit samples and 83 male kiwifruit samples were ...

Embodiment 3

[0057] Example 3 Conversion of Specific Molecular Markers and Establishment of Fluorescent Capillary Detection Molecular Marker System

[0058] (1) Establishment of multiplex PCR system

[0059] FAM fluorescent groups were added to the 5' ends of Ms1, Ms2 and ITS primers, and three corresponding pairs of FAM fluorescent primers were synthesized. The three pairs of primers were mixed into one PCR reaction, and the annealing temperature and primer ratio of the primer reaction were adjusted to construct The multiple PCR reaction system can amplify 3 sites in the same reaction system, and the yield of the final product is equivalent. The following table is the PCR amplification system we obtained after optimization.

[0060]Table 3: PCR amplification system and program

[0061]

[0062] In order to ensure the concentration balance of different products in the same reaction system, we adjusted the ratio of three pairs of primers in the reaction system according to the differenc...

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Abstract

The invention provides kiwi fruit sex specific molecular marker primers based on a fluorescent capillary electrophoresis detection technology and application thereof, and belongs to the technical field of molecular biology. According to the specific molecular marker primers, three loci can be amplified in the same PCR reaction system, the three loci comprise two male specific loci and one genomic DNA internal reference ITS locus, and the three loci are simultaneously detected in fluorescent capillary electrophoresis detection, so that the reliability of a detection system is greatly improved, and the detection cost is reduced.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a group of kiwifruit sex-specific molecular marker primers based on fluorescent capillary electrophoresis detection technology and applications thereof. Background technique [0002] The development of kiwifruit industry originated in the early 20th century, and the development of kiwifruit industry in my country began in the late 1970s. The varieties cultivated in the early days mainly came from Actinidia chinensis Planch.var.chinensis and A.chinensis Planch.var.deliciosa, which are also the main species of kiwifruit commercially cultivated at home and abroad. [0003] The flower types of Actinidia plants are complete flowers, and most of them are functional dioecious plants, which show that the stamens of the female flowers are aborted without producing vigorous pollen, and the pistils of the male flowers are aborted without forming ovules. In cultivation...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6879C12Q1/686C12N15/11
CPCC12Q1/6895C12Q1/6879C12Q1/686C12Q2600/16C12Q2600/166C12Q2565/125C12Q2563/107C12Q2537/143
Inventor 方林川戢小梅陈志伟张鸿翟敬华
Owner WUHAN ACADEMY OF AGRI SCI
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