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Molecular marker system for identifying authenticity of sugarcane hybrid and development method of molecular marker system

A technology of molecular markers and authenticity, applied in the fields of bioinformatics and molecular genetic breeding, can solve the problems of limited number of markers, missed screening, and small number of markers, etc., achieve stable amplification effect, facilitate popularization and application, and reduce missed detection rate Effect

Active Publication Date: 2021-11-02
NANTONG UNIVERSITY
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

There are three main problems with this method: First, the number of markers available is very limited
The sugarcane genome is very complex, and the development of molecular markers is progressing slowly. The markers currently used are mainly derived from molecular markers of related species such as sorghum. Therefore, it is difficult to obtain a sufficient number of polymorphic markers between parents
Second, the authenticity identification process of hybrids is seriously missed
Due to the small number of markers, especially the uneven distribution of markers, that is, the acquired markers may only come from a small number of chromosome intervals, which will lead to serious missed screening in the process of hybrid authenticity identification
For example, due to recombination, the offspring of the hybrid cannot carry the chromosome covered by the detection marker, it will be considered as a false hybrid and eliminated, resulting in missed detection
The third is that the existing markers have poor applicability and cannot be used as a general marker for hybrid authenticity
This is mainly due to the fact that the markers have not been screened within the species and are not conserved within the species.
These problems seriously restrict the development of sugarcane hybrid seed production. Therefore, it is urgent to develop markers for the two parental species of modern sugarcane cultivars, and establish an interspecific polymorphic marker system that is conserved within the species and covers different chromosomes.

Method used

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  • Molecular marker system for identifying authenticity of sugarcane hybrid and development method of molecular marker system
  • Molecular marker system for identifying authenticity of sugarcane hybrid and development method of molecular marker system
  • Molecular marker system for identifying authenticity of sugarcane hybrid and development method of molecular marker system

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Example 1: Homologous differential DNA sequence acquisition of tropical species and cut hands dense species

[0024] Use the RepeatMasker software to filter the repetitive sequences in the genomes of tropical species and cut-hand dense species, and then break the genome into 50bp DNA sequences at intervals of 5 bases, and paste these short sequences into the genome to further verify the sequence Genome source. The method of BLAST sequence alignment of short sequences from two genome sources is compared with each other to obtain homologous and differential DNA sequences.

[0025] The invention uses whole genome sequence information, uses RepeatMasker software to filter repeated sequences, and obtains difference DNA sequence information at the whole genome level between two sugarcane species through mutual comparison.

[0026] It should be noted that the RepeatMasker software used here for filtering repetitive sequences and BLAST sequence alignment is a prior art, and it...

Embodiment 2

[0027] Example 2: Primer Design and PCR Amplification of Differential DNA Sequences

[0028] The primer design software Primer Premier 5.0 was used to design primers for the differential DNA sequences. The length of the primers was required to be 20 ± 5 bp when designing the primers, and the primers should be designed in the conserved regions at both ends of the differential sequences.

[0029] The reaction system of PCR is: 0.25 μL each of primers, 2 μL of DNA template, 5 μL of PCRMix, and finally add ddH2O to make up the total volume to 10 μL.

[0030] The conditions of PCR amplification were: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 61°C for 30 s, extension at 72°C for 60 s, 28 cycles, and final extension at 72°C for 10 min.

Embodiment 3

[0031] Embodiment 3: the electrophoresis detection of PCR product

[0032] The electrophoresis that the present invention adopts is polyacrylamide electrophoresis, and flow process is:

[0033] 1. After installing the glue tank, configure polyacrylamide gel working solution (8% polyacrylamide, 300ul10% AP, 60ul TEMED), and pour it into the glue tank.

[0034] 2. After the gel is solidified, add 1×TBE electrophoresis buffer (10.8g Tris, 5.5g boric acid, 4mL EDTA (0.5M, pH 8.0), 80ml ultrapure water, dilute to 100mL).

[0035] 3. Add 2ul of the PCR product into the loading buffer, and take 1ul and put it into the prepared gel spotting well.

[0036] 4. Use 180v constant voltage for electrophoresis for 100 minutes.

[0037] 5. End the electrophoresis, remove the gel and put it into the dye solution (1L of pure water is dissolved by adding 1g of silver nitrate), and rotate horizontally on the shaker for 15min.

[0038] 6. Wash the gel twice with ultrapure water, 30s each time. ...

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Abstract

The invention relates to the technical field of bioinformatics and molecular genetic breeding, in particular to a molecular marker system for identifying the authenticity of sugarcane hybrids and a development method of the molecular marker system. Whole genome DNA sequences of two parents of a modern sugarcane cultivar, i.e., Saccharum Spontaneum species and tropical species, are compared, homologous sequences with interspecific differences are selected, molecular marker primers aredesigned, PCR amplification and electrophoresis detection are carried out by adopting the plurality of tropical species and Saccharum Spontaneum species, molecular markers covering eight interspecific polymorphisms of different chromosomes are selected, and a heterozygous band type is generated in a hybrid F1 by the marker system. The polymorphic marker between the tropical species and the Saccharum Spontaneum species can be used for detecting the authenticity of sugarcane hybrids, the miss detection rate of real hybrids can be greatly reduced due to the fact that markers cover all chromosomes, meanwhile, each marker has high conservative property in the species, and the markers can be used for identifying the authenticity of hybrid progeny between different parent combinations and has good general applicability.

Description

technical field [0001] The invention relates to the technical fields of bioinformatics and molecular genetic breeding, in particular to a molecular marker system for authenticity identification of sugarcane hybrids and a development method thereof. Background technique [0002] Sugarcane is the most important sugar and energy crop in the world, and its sucrose production accounts for more than 80% of the world's sucrose production. Modern sugarcane cultivars are mainly hybrids of tropical species with high sugar content and stress-resistant close species of cut hands. However, both the tropical species and the cutshoud species are polyploid plants with complex genomes. The tropical species is an octaploid with 80 chromosomes (2n=8x=80); the genome of the cut-handed species is more complex than that of the tropical species, and its different materials (clone, clone) often have different chromosome compositions. At present, up to nearly 40 different types of chromosomal numb...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888C12Q2600/156C12Q2600/13
Inventor 王凯禄小溪张会
Owner NANTONG UNIVERSITY
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