RAPD Analysis Of Genetic Diversity And Indentification Of Molecular Markers Linked To Cytoplasmic Male-Sterility Ioci In Allium Cepa

Onion(Allium cepa L.) is wildly cultivated in the world and its production area posses the third ranks in all of the vegetables.It is as a very important vegetable for the domestic cultivation and exporting to get foreign currency in China.However,the F1 hybrid seeds are primarily depended on import from other countries,it causes farmers have to pay high cost for the onion production.On the other hand,onion plant has great heterosis plant,therefore,the F1 hybrid varieties are the first priority for the recent onion breeding programs.But it needs long period to breed a hybrid onion variety by using traditional way,because onion species belong to the biennial plant.In order to establish a effective breeding system to breed F1 hybrid onion,the first,fifty different germplasm were collected and the genetic diversity of those resources was analyzed with RAPD,the second,identified the sterile-type or fertile-type of cytoplasm by using molecular markers,the results are as follows:The high-quality genomic DNA was obtained by the CTAB method.The optimal PCR system for RAPD analysis was as follows:14.17μL ddH₂O,150μmol/L dNTP, 2.0μmol/L Mg²⁺,2.5μL Buffer,0.5μmol/L random primer,20 ng template,1.5 U Taq polymemse in 25μL reaction system.The program of amplifying reaction was as follows:After pre-denaturing at 94℃for 5 min,under the condition of denaturing at 94℃for 1 min,annealing at 36℃for 1 min,extension at 72℃for 1.5 min, amplifying for 45cycles,and extension at 72℃for 7 min at last.The cluster analysis among 50 onion accessions showed that 33 polymorphic sites were detected among the total 81 sites amplified by 10 screened random primers (among 400 primers).The genetic distance(GD) of 50 onions was 0.042～0.269,the germplasm could be classified into 6 groups at 0.168 of GD,it indicates that the genetic basis of them was rather narrow.The primer sequence was designed according to the literatures and the cytoplasmic male sterility line S110,S118 and maintainer line N210,N218 was amplified by PCR. It was shown there was a 339 bp specific band in male sterility line S110 and S118, but not found in maintainer line N210 and N218.The confirmation test of this molecular marker,more than 10 sets of male sterile line and maintainer line were provided.All phenotype of these lines were confirmed betbre this test by checking the character of expressions whether possessing normal pollen or not.This result showed that the molecular marker is completely same with visual markers.It conclude that this molecular marker is very useful for distinguish sterile-type or fertile-type of cytoplasm in breeding program.