| Vernalization and photoperiod response are important traits of wheat. They are direct influenced the wheat growing areas, seed introduction and cultivation techniques, and it's also have great impact on yield, disease resistance, stress resistance, and many other important agronomic traits. So the study on vernalization and photoperiod are very important to seed introduction, breeding and management of cultivation. Ecotilling is a high throughput reverse genetic research method for detecting single nucleotide polymorphism (SNP) in the genes of a natural population; it can identify mutation sites rapidly in the target gene of natural population. Therefore, using Ecotilling to analyze vernalization gene and photoperiod gene can help us understanding the molecular regulation of flowering and the alleles of wheat, and it can also give us references on improving growth-period trait that based on molecular breeding.In our research, we optimized the traditional Ecotilling technique, using agarose gel electrophoresis to detect the digestion fragments, and achieved better results. We detect the partial coding region of vernalization gene VRN-A1and photoperiod gene Ppd-D1 in some wheat materials, and analyzed the relationship between the SNPs and the corresponding phenotype. The main results were summarized as follows:(1) Optimized the traditional Ecotilling technique, using positive plasmids which have base mismatch to create heteroduplex, then we detect the digestion fragment by agarose gel electrophoresis successfully. However we established Ecotilling that based on agarose gel electrophoresis basically.(2) We detect SNP in wheat vernalization gene VRN-A1 using agarose gel electrophoresis as an example to determine the optimal parameters in wheat Ecotilling. The result shows that the best amount of template used in PCR was 160 ng, the best ratio is 1 : 1 used for DNA pooling, enzyme amount used in digestion is 2.4μl, the digestion time is 45min, and 2% agarose gel is also the best choice. Therefore, an efficient method for detecting SNP in genes of polyploidy materials such as wheat was established by optimizing the Ecotilling procedure and the agarose gel electrophoresis detection.SNPs in the VRN-A1 gene associated with vernalization among 108 wheat accessions were detected using agarose gel Ecotilling. Based on the SNPs detected, these accessions were found to fall into different haplotype categories, namely, A, B, and C, each with five, six, and three variations, respectively. The sequencing analysis showed that the variations included conversion, transversion, and deletion of a single base, as well as the insertion of a small fragment. At the same time, the winter phenotype was found to be mainly determined by haplotype A and B, while the spring phenotype was mainly determined by haplotype C. Three SNPs are considered associate with wheat vernalization and another three mutations'function is unsure.(3) We used Ecotilling that based on agarose gels to screen SNPs of key coding regions of photoperiod gene Ppd-D1 in 108 wheat varieties. the results showed that there were three specific digestion bands for the landrace Hongmangmai,Hongmangmai2 and Ningchun27, the sequencing result indicated that there exited two SNPs (C/T;G/A) in the target region compared with the control. However, Banong1, Dingxi35, QW6285 and other four materials have two specific digestion bands, and their target region have a 5bp deletion compared with the control. Further analysis revealed there was no clear correlation between the SNPs in Ppd-D1 with the photoperiod of wheat, the influence of the SNPs on heading date were to be further clarified; the sequences of the remaining varieties are identical to the control.
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