Application of a short DNA sequence in the identification of citrus taxa and citrus seedlings

A short-sequence, citrus technology, applied in the field of genetic engineering, can solve problems such as low overall cost, and achieve the effects of high specificity, high reliability and good repeatability

Active Publication Date: 2021-10-29
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, the current gene cloning and sequencing technology is relatively mature. The early cloning and post-analysis can be carried out by general laboratories, and the intermediate sequencing has many commercial companies to choose from. The overall cost of a single sample is relatively low, and the identification time is also short. faster

Method used

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  • Application of a short DNA sequence in the identification of citrus taxa and citrus seedlings
  • Application of a short DNA sequence in the identification of citrus taxa and citrus seedlings
  • Application of a short DNA sequence in the identification of citrus taxa and citrus seedlings

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Short sequence analysis of Wanjincheng

[0044] 1. The synthesis of forward and reverse primers used to amplify this short sequence requires acrylamide gel electrophoresis purification grade.

[0045] 2. Genomic DNA extraction of Wanjincheng. Genomic DNA of Wanjincheng was extracted by the improved CTAB method. The concentration of the extracted DNA was detected by a micro-volume ultraviolet spectrophotometer, the integrity of the extracted DNA was detected by 1% agarose gel electrophoresis, and it was stored at 4°C for future use.

[0046] 3. DNA polymerase chain reaction (PCR) amplification to obtain the target short sequence. The PCR system is as follows: 1×PCR Buffer, 1.5mmol L -1 Mg 2+ , 0.2mmol·L-1 of dNTPs, 0.33nmol·L -1 The upstream and downstream primers, 1U of Takara high-fidelity Taq DNA polymerase, about 100ng of DNA template, and a total volume of 50μL. The amplification program is: pre-denaturation at 94°C for 4 min; denaturation at 94°C f...

Embodiment 2

[0053] Example 2: Short sequence analysis of Oita Satsuma

[0054] 1. The synthesis of forward and reverse primers used to amplify this short sequence requires acrylamide gel electrophoresis purification grade.

[0055] 2. Genomic DNA extraction of Oita Satsuma. Genomic DNA of Oita Satsuma mandarin was extracted by the improved CTAB method. The concentration of the extracted DNA was detected by a micro-volume ultraviolet spectrophotometer, the integrity of the extracted DNA was detected by 1% agarose gel electrophoresis, and it was stored at 4°C for future use.

[0056] 3. DNA polymerase chain reaction (PCR) amplification to obtain the target short sequence. The PCR system is as follows: 1×PCR Buffer, 1.5mmol L -1 Mg 2+ , 0.2mmol·L-1 of dNTPs, 0.33nmol·L -1 The upstream and downstream primers, 1U of Takara high-fidelity Taq DNA polymerase, about 100ng of DNA template, and a total volume of 50μL. The amplification program is: pre-denaturation at 94°C for 4 min; denaturati...

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Abstract

The invention belongs to the technical field of genetic engineering, and discloses the application of a short DNA sequence in the identification of citrus groups and citrus seedlings. The primer sequences for identifying the short DNA sequence are SEQ ID NO: 1 and SEQ ID NO: 2, and are composed of The sequences defined by the primers are in the representative sequences of the main citrus groups and their SNP, SSR and Indel combinations. The present invention amplifies the citrus genome with given primers to obtain target short sequences and perform cloning and sequencing analysis; according to the variation characteristics or combination of fragment SSR, Indel and / or SNP, the heterozygous state of citrus, citrus groups and Identification of citrus seedlings. The invention completes the analysis and identification of different materials of citrus fruit trees by cloning and sequencing a specific short sequence and comparing the characteristics of the sequencing sequence. Classification of citrus fruit trees was carried out by genome amplification, cloning, sequencing, and comparison analysis with the combination feature table.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and relates to the application of a short DNA sequence in the identification of citrus groups and citrus seedlings. Background technique [0002] Citrus fruit trees belong to Rutaceae. Due to the influence of apomixis, easy hybridization between genera and species, asexual variation, extensive cultivation, and long history, citrus fruit trees have produced rich variations. Tangelo, citron, lemon, lime and orange lime, wild and semi-wild Yichang orange, etc., as well as trifoliate trifoliate and its hybrids, which are mainly used as rootstocks, are mainly distributed in Australia. and Oxalmon etc. The research on citrus taxonomy and phylogenetic evolution plays an important role in clarifying the genetic and evolutionary relationship of citrus fruit trees, better understanding and development and utilization of citrus fruit trees, carrying out citrus genetic breeding and further collec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/6895C12Q1/6858
CPCC12Q1/6858C12Q1/6895C12Q2600/156C12Q2531/113C12Q2535/122C12Q2565/125
Inventor 洪棋斌龚桂芝
Owner SOUTHWEST UNIV
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