Simple sequence repeat-polymerase chain reaction (SSR-PCR)-based hybrid rape seed purity detection method

Abstract

The invention discloses a simple sequence repeat-polymerase chain reaction (SSR-PCR)-based hybrid rape seed purity identification method, which comprises the following steps of: performing sprouting culture on hybrid rape sample seeds to be detected, performing alkaline lysis on the cultured seedlings, simultaneously performing ultrasonic disruption treatment, and adding an extracting buffer solution to obtain a genome DNA solution; performing PCR amplification on genome DNA by using an SSR primer sequence; performing voltage stabilizing electrophoretic separation on a PCR amplification product in agarose gel; performing imaging and tape reading on the PCR amplification product subjected to electrophoretic separation in a gel imaging system, comparing band characteristics of the sample seeds with those of parent seeds, counting seeds with the band characteristics of male parent and the band characteristics of female parent in the sample seeds, and obtaining the purity of the hybrid rape seeds to be detected according to a variety purity formula. The identification method has the advantages of quickness, simplicity, convenience, high throughput, low detection cost, high detection efficiency, stable and reliable detection results and the like.

Description

technical field [0001] The invention belongs to the technical field of rapeseed breeding and application, and in particular relates to a method for detecting the purity of rapeseed hybrid seeds. Background technique [0002] Brassica napus has obvious superparent vigor, and is one of the crops with the most successful use of heterosis. Since my country began to popularize and apply the first Brassica napus hybrid variety Qinyou No. 2 in the 1980s, the hybrid variety has been popularized rapidly, which has greatly improved the production level of rapeseed in our country. However, at present, the pol-CMS system is generally used in the hybrid seed production of rapeseed. The female sterile line of its parent is low temperature sensitive. The purity and quality decline, and in serious cases, it can cause a substantial reduction in the production of rapeseed. Therefore, strict purity identification must be carried out before rapeseed hybrids are used in production, especially ...

AI Technical Summary

Problems solved by technology

However, the detection cost of this type of marker technology is relatively high, and the amount of genetic information is very limited, and there is no difference between the parents of most hybrid rapeseed, which determines that it can only be applied to the purity detection of a few rapeseed hybrids; in recent years Recently, the rapid development of molecular marker technology has opened up a new way for the purity detection of rapeseed hybrids, especially the SSR marker technology, which is not only relatively simple to operate, but also has a wealth of genetic information. In theory, as long as suitable primers are screened, It can be applied to the purity detection of any rapeseed variety, which has become the preferred molecular marker technology for the purity detection of rapeseed hybrids, and has been applied in some universities and scientific research units

[0004] However, the existing SSR-PCR purity detection method for rapeseed hybrids has cumbersome operation steps, consumes a lot of manpower and time, and requires personnel with certain experimental skills to operate; on the other hand, the instruments, reagents and consumables required by this method are very expensive. It is difficult for ordinary-scale seed companies to build a testing system and bear testing costs

Therefore, the existing SSR marker detection method is not suitable for the current large-scale commercial hybrid seed production purity detection needs, and it is even less likely to be widely used in small seed enterprises

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