Method for rapidly detecting polymorphism of sugarcane sucrose phosphate synthase B (SPSB) gene and application of method

A technology of gene polymorphism, SPSB2177P1, which is applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems that the intron region has not been cloned, and the polymorphism analysis of the intron region has not been reported. , to overcome the effects of high cost, high accuracy and strong primer specificity

CN104561365AInactive Publication Date: 2015-04-29SUGARCANE RES INST OF YUNNAN ACADEMY OF AGRI SCI
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Publication Date
2015-04-29
Estimated Expiration
Not applicable · inactive patent

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Abstract

The invention discloses a method for rapidly detecting the polymorphism of a sugarcane sucrose phosphate synthase B (SPSB) gene. The method comprises the steps of designing a pair of primers to carry out PCR amplification by taking the whole-genome DNA of saccharum robustum B.51NG3, saccharum species ROC22, saccharum.officinarum L.Badila, saccharum sinense R. lustrous bamboocane, saccharum barberi J.Nagori or saccharum spontaneum L.yinge No.1 as a template; and carrying out agarose gel detection to name the SPSB genotype of a fragment with the size of 175bp as type B1, name the SPSB genotype of a fragment with the size of 294bp as type B2 and name the SPSB genotype of a fragment with the size of 421bp as type B3. The invention provides a DNA level identifying method for saccharum sugar trait marker-assisted selection, and the DNA level identifying method is beneficial to tracking of different haplotypes of SPSB genes in filial generations of saccharum and has important significance for rapidly establishing saccharum germplasm with excellent heredity.
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Description

technical field

[0001] The invention belongs to the technical field of plant molecular breeding, and in particular relates to a method for detecting the polymorphism of the SPSB gene (JN584485) of the genus Saccharum, and its application in marker-assisted selective breeding of sugarcane sugar traits to quickly establish genetically excellent sugarcane germplasm. Background technique

[0002] Sugarcane is a complex allopolyploid plant with multiple sets of chromosomes in the nucleus. In the cross-breeding of sugarcane, the tropical species of Sugarcane (Saccharum. ) (Saccharum spontaneum L.), Saccharum robustum B., Saccharum sinense R. and Saccharum barberi J. were mainly interspecific hybrids. Therefore, the traits of hybrid progeny are seriously separated, and the progeny of a hybrid panicle has hundreds of progeny types, and the transmission mode of each haplotype in the progeny cannot be precise. Sucrose phosphate synthase (SPS) is one of the most critical enzymes in su...

Examples

Embodiment 2

[0071] (1) Cloning of partial DNA sequence of sugarcane SPSB gene and detection of polymorphism.

[0072] 1. Extraction of sample DNA

[0073] Saccharum robustum B. 51NG3 was used as the detection object, and the genomic DNA was extracted with the EasyPure Plant Genomic DNA Kit provided by Quanshijin Company, and finally dissolved in sterilized ultrapure water, and the detection was carried out at -20°C save. The OD value of the DNA sample at 260nm and 280nm was measured with a UV photometer. Calculate the DNA content and the ratio of OD260 / OD280. If the ratio of OD260 / OD280 is less than 1.6, it means that the sample contains more protein or phenol, and purification should be performed; if the ratio is greater than 1.8, RNA purification should be considered. After the DNA detection is completed, a certain amount is taken out and diluted to 40ng / μL for use as a PCR template.

[0074] 2. Primer design

[0075] Obtain the published SPSB gene sequence from NCBI, GenBank ID is ...

Embodiment 3

[0096] (1) Cloning of partial DNA sequence of sugarcane SPSB gene and detection of polymorphism.

[0097] 1. Extraction of sample DNA

[0098] Saccharum robustum B. 51NG3 was used as the detection object, and the genomic DNA was extracted with the EasyPure Plant Genomic DNA Kit provided by Quanshijin Company, and finally dissolved in sterilized ultrapure water, and the detection was carried out at -20°C save. The OD values ​​of DNA samples at 260nm and 280nm were measured with a UV spectrophotometer. Calculate the DNA content and the ratio of OD260 / OD280. If the ratio of OD260 / OD280 is less than 1.6, it means that the sample contains more protein or phenol, and purification should be performed; if the ratio is greater than 1.8, RNA purification should be considered. After the DNA detection is completed, a certain amount is taken out and diluted to 40ng / μL for use as a PCR template.

[0099] 2. Primer design

[0100] Obtain the published SPSB gene sequence from NCBI, GenBan...