Functional marker for paddy thermo-sensitive male sterile gene tms 5 and application of functional marker

Abstract

The invention provides a functional marker for a paddy thermo-sensitive male sterile gene tms5, a method of testing whether a sample contains a tms5 gene or not by using the functional marker, a method of judging the seed purity of two-line thermo-sensitive paddy by using the functional marker, and the application of the functional marker in cultivating a sterile line. According to a mutation site sequence of the paddy tms5 gene, a mismatched base is introduced into a PCR primer, so that a paddy PCR amplification product containing a tms5 gene (mutation type) generates an enzyme cutting site of a restriction enzyme Rsa I, while a paddy PCR product not containing a tms5 gene (wild type) does not have the enzyme digestion site. The PCR product is subjected to enzyme digestion by the Rsa I, and the paddy PCR amplification product containing the tms5 gene is turned into two segments by enzyme digestion; but the paddy PCR product not containing the tms5 gene cannot be digested; thus distinguishing can be performed. By adopting the design that the tms5 amplification product is directly subjected to enzyme digestion, compared with the prior art, on one hand, the detection process is simplified, and on the other hand, the occurrence of a false positive result is avoided.

Description

technical field [0001] The invention belongs to the field of rice molecular detection. Specifically, the present invention discloses a functional marker designed for the tms5 functional mutation site, and the functional marker can be used to detect whether a rice sample contains the tms5 gene. Background technique [0002] The discovery and successful application of two-line sterile rice resources have played an important role in ensuring food security in my country. Compared with the three-line sterile line, the two-line hybrid rice is not restricted by the restorer gene, can be combined freely, and has a high resource utilization rate, which makes the yield and rice quality of the two-line hybrid rice significantly improved compared with the three-line hybrid rice . At the same time, the two-line CMS also has the advantage of self-propagation. Therefore, in recent years, the two-line hybrid rice has developed rapidly in my country, and the planting area and promotion ra...

AI Technical Summary

Problems solved by technology

The amplified products of RZ2F1 / R and RZ2F2 / R are both 178bp, but the Sty I restriction site is introduced into the RZ2F1 / R product, which can cut the amplified product (153bp and 25bp) with 70-72bp position as GCG, while The amplified product of the tms5 gene (70-72bp is TAG) cannot be cut. The Hinf I enzyme cutting site is introduced into the RZ2F2 / R product, which can cut the amplified product of the 70-72bp position of TCG, and also cannot cut tms5 gene amplification product

This design uses two amplifications and two enzyme digestions to infer whether the tms5 gene is contained according to the enzyme digestion results. The process is cumbersome. TAG three haplotypes, but it does not rule out the possibility of other haplotypes at the 6-base restriction site of HinfI and StyI. If there are other haplotypes, HinfI and StyI cannot be cut, which will lead to false positive results

[0011] Therefore, to sum up, at present, for the verification of the rice temperature-sensitive sterile gene tms5, the verification methods adopted either have false positives in the verification of the gene, or require two enzyme digestion verifications before it can be verified, and even Through two enzyme digestions, for some specific cases, the appearance of false positive results cannot be avoided

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