Construction Of A Molecular Genetic Linkage Map Of Peanut

Peanut is an important cash crop in the world. It is an important source for food protein and vegetable oil in china, and plays an inportant pole in the national economy. Conventional breeding methods did not meet the requirements of emerging problems in developing peanut varieties with high yield and good quality and resistance/tolerance to biotic and abiotic stresses. The main trend in peanut breeding is advanced molecular marker-assisted selection (MAS) breeding technology. Construction of high-density genetic linkage map could provide technical information for improving the important agronomic traits of peanut, and it is the basis and guarantee of studying complex traits at the molecular level. The rapid development of DNA molecular marker technology provides more marker types for construction of peanut genetic linkage map. In this study, a genetic linkage map of peanut was constructed by means of molecular markers including SSR (microsatellites or simple sequence repeat), AhTE (Arachis hypogaea transposable element), ISSR (inter simple sequence repeat), SRAP (sequence-related amplification polymorphism) and SRAP-RGA (sequence-related amplification polymorphism-resistance gene analog), the results were as follows:1. Optimization of DNA isolation:the method of CTAB that extracted total DNA of the peanut was modified and an optimal method for extracting DNA from the young leaves of the peanut was obtained.2. Optimization of SRAP-RGA amplification system:optimization of SRAP-RGA amplification system were undertaken including4factors of Mg2+, dNTP, annealing temperature and PAGE gel crosslinking and obtained an optimal SRAP-RGA amplification system for our sample.10μL PCR reaction mixture included1μL10×PCR buffer (Mg2+free),2.0mmol/L Mg2+,250μmol/L dNTPs,50ng DNA,0.5μmol/L Primer and0.5U Taq DNA polymerase. The thermal cycling program was as follows:5min denaturation at94℃,5cycles of1min at94℃,1min annealing at45℃,1min extension at72℃;30cycles of94℃1min,50℃1min,72℃1min; and a final10min extension at72℃.3. Screening of polymorphic primers:2parent of the mapping population, H22and D99, were screened with2171pairs of SSR primers,528pairs of AhTE primers,10pairs of ISSR primers,460pairs of SRAP primers, and507SRAP-RGA primers combination. As a result,211markers containing186SSR primers,8AhTE primers,1ISSR primers,8SRAP primers and8SRAP-RGA primers showed polymorphism between mapping parents and population.4. Construction of the linkage map:A genetic linkage map for the peanut was constructed using JoinMap4.0based on94F2individuals from H22×D99cross. It consisted of158markers, which distributed onto20linkage groups (LGs)(LOD≥3.0) and spanned a total map distance of1170.1cM with all average interval of7.41cM between markers. The maximum and minimum distance between two markers was73.5cM and0.2cM, respectively. Each linkage group ranged from6.8cM and251.2cM had2to28markers.