| Melon and watermelon F1 hybrid seeds, with significant heterosis of growth, high resistance to diseases and stress, well-setting fruit, high yield, good fruit quality and long storage period, were widely used in commercial production. However, at present, in the melon and watermelon hybrid seeds production, the emasculation and pollination were usually made by hand. It is hard to control the genetic purity of hybrid seeds. In addition, currently, more and more false and inferior seeds were used in the commercial trade. Because low genetic purity would cause big losses, it is necessary to develop a rapid, accurate and simple method to assess the genetic purity of hybrid seeds in melon and watermelon.In this study, the main factor influence the RAMP-PCR system including Mg2+, dNTPs and primers were optimized for genome DNA in melon ’Haimi 2’ and watermelon ’Sumi 5’. The optimization of RAMP-PCR system for these two cultivars was Mg2+ at 0.8-4.0 mmol·L-1, dNTPs at 0.08-0.4 mmol·L-1, primer at 0.08-0.32 μmol·L-1. For these two cultivars, the most suitable of RAMP-PCR system are primers at 0.32 μmol·L-1, dNTPs at 0.2 mmol·L-1, Mg2+ at 2.0 mmol·L-1, DNA at 10 ng and TaqE at 0.8U in a 20μL reaction. The analysis of the annealing temperature of the gradient PCR indicated that while the temperature ranged from 38 to 50℃, the obvious difference of production of RAMP reaction was observed. In which, the annealing temperature of 44℃ is suitable for RAMP-PCR in watermelon’Sumi 5’, while 43℃ is appropriate for RAMP-PCR in melon ’Haimi 2’.Three molecular marker systems, RAPD, SRAP and RAMP, were employed to test the genetic purity of melon F1 hybrid cultivar’Haimi 2’. A total of 358 primers, including 96 SRAP,118 RAMP and 144 RAPD primers, were amplified to screen the polymorphic loci between F1 hybrid cultivars and its parental lines. The result showed that six primers including two SRAP (em6/fc8 and em7/pm36), two RAMP (RP193/rm24 and RP538/ml) and two RAPD primers (RP204 and RP401) could generate female parent-specific makers (FPS) and male parent-specific markers (MPS) simultaneously in F1 hybrid individuals. These six effective primers were further used to investigate the genotype of 210 individuals, and six out of 210 F1 individuals were identified to be false hybrids with the primer em6/fc8, em7/pm36, RP193-rm24, RP204 and RP401. While with the primer RP538/ml, only five Fi individuals were identified to be false hybrids. This discrepancy may be due to the heterotic loci remained in the parental line. Therefore, it could be confirmed that these six individuals were false hybrids and consequently, the genetic purity of this seed was 97.1%. The results of our study were consistent with the field trials, indicating that the multiple molecular markers for Fi hybrid seed genetic purity testing were rapid and efficient with high accuracy. In order to identify genetic purity of ’Sumi 5’, two SRAP (eml/pm36, em7/sa20), two RAMP (RP560/N4, RP538/K46) and two RAPD primers (RP976, RP1077), which produced both female and male parent-specific markers, were selected from 300 RAPD primers,200 RAMP primer combinations and 152 SRAP primer combinations. We employed six primers on’Sumi 5’, and seven individuals were identified to be false hybrids of 210 F1 individuals and the genetic purity was 96.7%, which was consistent with field trials. The results indicated that the multiple molecular markers are effective and useful tools for identifing the seed purity of Fi hybrid seed in plants.This study indicated that the molecular marker systems are effective and useful tools for genetic purity testing of Fi hybrid seeds in melon and watermelon, which could replace the traditional method basing on morphological characteristic inspection. In addition, it is also noteworthy that several molecular markers could be comprehensive utilized to make the results more accurate and reliable. |
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