Codominant RFP molecular marker applicable for breeding citrus grandis with red-peel characteristic and application of codominant RFP molecular marker

A molecular marker, co-dominant technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of long breeding cycle, low efficiency, and single color, and achieve high nutritional value. , the effect of speeding up the color and reducing the workload of breeding

Active Publication Date: 2015-12-09
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Most of the traditional pomelo varieties have yellow or light yellow exocarp, which has a single color because they cannot accumulate anthocyanins on the pericarp. Traditional phenotypic selection methods introduce the feature of red skin into traditional pomelo varieties, and there are long breeding cycles, Low efficiency and other disadvantages

Method used

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  • Codominant RFP molecular marker applicable for breeding citrus grandis with red-peel characteristic and application of codominant RFP molecular marker
  • Codominant RFP molecular marker applicable for breeding citrus grandis with red-peel characteristic and application of codominant RFP molecular marker
  • Codominant RFP molecular marker applicable for breeding citrus grandis with red-peel characteristic and application of codominant RFP molecular marker

Examples

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Effect test

Embodiment 1

[0039] 1. Preparation of genomic DNA of citrus such as pomelo, citron, lemon, and red summer orange

[0040] In this example, the improved CTAB method was used to extract the genomic DNA of citrus such as pomelo.

[0041] 2. Utilize the primer pair RFPF / R1 and RFPF / R2 developed by the applicant to amplify and analyze the genomic DNA of all the above-mentioned citrus such as pomelo, citron, lime, and red summer orange.

[0042] The PCR reaction system is as follows: 2 μl PCR buffer, 2 μl MgCl 2 , 0.4dNTPs, TaqDNA polymerase (both purchased from Takara Company), 100ngDNA, each 0.2μM of forward and reverse primers, ddH 2 O supplemented to a final volume of 20ul. The thermal cycle parameters are: 95°C for 5min; 95°C for 30sec, 55°C for 45sec, 72°C for 45sec, 35 cycles; 72°C for 10min, 1 cycle; 4°C storage, the reaction is completed on the S1000ThermalCyclerPCR instrument. The amplified product was detected by 1.0% agarose gel electrophoresis on a horizontal electrophoresis tank...

Embodiment 2

[0044]Example 2 Screening and Identification of the Hybrid Offspring of Red Pomelo and Guanxi Honey Pomelo

[0045] 1. Preparation of genomic DNA of about 200 strains of red skin pomelo, Guanxi honey pomelo and their hybrid offspring

[0046] In this example, the improved CTAB method was used to extract the genomic DNA of 200 strains of Red Pimelo, Guanxi Honey Pomelo and their hybrid offspring.

[0047] 2. Using the primer pair RFPF / R2 developed by the applicant to amplify and analyze the genomic DNA of red skin pomelo, Guanxi honey pomelo and their hybrid offspring

[0048] The PCR reaction system is as follows: 2 μl PCR buffer, 2 μl MgCl 2 , 0.4dNTPs, TaqDNA polymerase (both purchased from Takara Company), 100ngDNA, forward and reverse primers 0.2μM each, ddH2O supplemented to a final volume of 20μl. The thermal cycle parameters are: 95°C for 5min; 95°C for 30sec, 55°C for 45sec, 72°C for 45sec, 35 cycles; 72°C for 10min, 1 cycle; 4°C storage, the reaction is completed on...

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Abstract

The invention belongs to the technical field of molecular breeding of citrus, and particularly relates to a codominant RFP molecular marker applicable for breeding citrus grandis with a red-peel characteristic and application of the codominant RFP molecular marker. Most varieties of citrus grandis are light yellow in peel and single in color in China; the citrus grandis with the red-peel characteristic is novel in color and variety, thereby being more attractive to consumers. The method includes: starting from a unique material, the red-peel citrus grandis, using a primer pair MYB2F/R to amplify the red-peel citrus grandis and common citrus grandis for regulating promoters of anthocyanin synthesis key transcription factor CgMYB2 genes so as to obtain two DNA fragments, then subjecting DNA polymorphic regions to PCR amplification, cloning and sequencing, and comparing two obtained nucleotide sequences to find that one segment of insertion mutation of 162bp is between the two sequences, resulting in polymorphism of the sequence-characterized amplified region; designing primer pairs RFP F/R1 and RFP F/R2 according to differences of the two nucleotide sequences prior to PCR amplification to obtain the codominant RFP molecular marker capable of distinguishing the red-peel citrus grandis from the common citrus grandis (being light yellow in peel). The invention further discloses a method for preparing the molecular marker.

Description

technical field [0001] The invention relates to the technical field of citrus molecular breeding, in particular to a pomelo co-dominant RFP molecular marker suitable for breeding red-skin traits and its application. The new genetically stable pomelo cultivars provide new molecular markers. Background technique [0002] Grapefruit (Citrusgrandis) is a citrus fruit of the Rutaceae family. It tastes sweet and sour and has high nutritional value. It is rich in protein, vitamins, calcium, phosphorus, iron and other essential elements for human body. Grapefruit has the functions of strengthening the spleen and stomach, nourishing the lungs, and helping digestion, and is very popular among people. my country is rich in pomelo resources, and most of the more common pomelo varieties in the market now have yellow or light yellow exocarp with a single color. However, red pomelo is a mutant of peel color, and the skin of its mature fruit is red. Red pomelo is a rare resource in pomelo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q2600/13C12Q2600/156
Inventor 徐强黄鼎温少华邓秀新
Owner HUAZHONG AGRI UNIV
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