| Pepper (Capsicum annuum L.) is widely cultivated vegetable in the word, which covers a bigger cultivation areas in China. Pepper is a cross-breeding vegetable, and the genetic purity of the hybrids is difficult to control due to the artificial factors during the breeding programs. Although morphological characters have been used for hybrids genetic purity identification in vegetables, the method exhibit time-consuming, laborious, less effective, and is susceptible to seasonal effects. Therefore, fast, accurate and simple detection of genetic purity of seed quality testing work has become an important issue, and it is necessary to develop an efficient identification of the genetic purity of hybrid seeds technology. In this study, we used RAMP, RAPD, ISSR, SSR and SRAP molecular markers to test the genetic purity of pepper, and analyzed the fingerprint of 24 pepper varieties. Our studies will provide technical support for detecting the genetic purity of hybrid pepper seeds. These results were summarized as follows:1. In this study, the main factors influence the ISSR system including Mg2+, dNTPs and primers were optimized for genome DNA in’Suzi No.1’. Each of them had 3 concentrations, with Mg2+ for 1.0,2.5,3.5 mmol-L-1,dNTPs for 0.1,0.15,0.25 mmol-L-1, primers for 0.1,0.4,0.6 μmol-L-1. The most optimized ISSR PCR system for 20μl is Mg2+ 2.5 mmol·L-1, dNTPs 0.15 mmol·L-1, primer for each 0.4μmol·L-1, DNA 10 ng, Taq DNA polymerase 0.8U. There were marked differences in PCR annealing temperature from 52.0 ℃ to 64.0℃, and it suggested that the best annealing temperature was 53.5℃.2. Using the hybrid’Suzi No.1’and the parents, by the means of RAMP, RAPD and SSR molecular marker-assisted selection system to identify the genetic purity of the DNA genomic.5(pairs) primers were selected out that presented co-dominance parents’ specificity bands, which included 1 pair RAMP primers, NAUK33/RP714,2 RAPD primers, NAURP570 and NAURP572,2 SSR primers, NAUSSR45 and NAUSSR9. With these 5(pairs) primers,210’Suzi No.l’F1 individuals were screened, and 3 individuals were elected for their female parent dominance only, and 2 for male only. Thence these 5 individuals were identified pseudohybrids. And the result was in accordance with the identification of the yield. Together, the molecular marker assisted technique could identify the genetic purity of’Suzi No.1’hybrid seeds.3. By combining genomic DNA and techniques of RAPD, SRAP, ISSR, and RAMP, we analyzed 24 species by the fingerprint methods containing four molecular markers digital fingerprints for Capsicum annuum L. The result suggested that there were no notable differences between species on the molecular level. To the four molecular marker assisted breeding (MMAB) methods, any one (pair) primer alone couldn’t distinguish each individual totally. The highest detectability primers, RP704 and IS35 just could make a distinction between 10 species. The cluster analysis also unveiled that MMAB could tell the similarity of horticulture traits and genetic relationship between species. A cultivar identification diagram (MCID) of the 24 pepper cultivars was constructed using polymorphic bands from the DNA fingerprints. The MCID method could separate all the cultivars from each other, with the polymorphic bands employed for identifying the cultivars and the corresponding primers being marked in the correct position on the MCID. |
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