Study On CDNA-AFLP Marker Linked To Leaf Color Trait In Mustard With Red Leaf

Mustard(Brassica juncea Coss.)is a kind of important cultivated vegetables and oil crops with strong economic worth.Red mustard is a special variety,it's famous for red leaf.The study on the mechanism of red leaf is not definitude.It will be propitious to create excellent germplasm resources,increase genetic diversity of species,breed better new varieties of ornamental and ediblevegetables;on the other hand,if the gene which close linked to leaf color trait in mustard can bemarked,then construct genetic map,it will lay the foundation on integrated genetic linkage map,and provide evidence for marker-assisted selection(MAS).An cDNA-AFLP systerm was established by red leaf character of mustard,select the cDNA-AFLP marker which close linked to leaf color traitin mustard,then analysis homology and annotate gene function.The study will promote further study on molecular lever of genetic basis in mustard,enrich genetic map on mustard,lay the foundation on marker-assisted selection,cloning and application of the gene which close linked to leaf color trait,improve competitive ability on mustard breeding of our country.The main results are as follows:1.Established a fit mustard cDNA-AFLP analysis systemIn this study,we select the red mustard which has uniqueness on leaf color as experiment material, extracting total RNA of different stage sample,using replacement synthesis method composed scDNA. Being digested by Taq I/Vsp I enzymes and ligated with synthetic adapter,we set up the systems of cDNA-AFLP analysis system.The main factors influencing cDNA-AFLP selective amplification were optimized on mustard.The cDNA-AFLP protocol system of mustard was established,that was:In 20μL PCR reaction,1.2mmol/L Mg²⁺,0.2 mmol/LdNTP,Taq DNA polymerase 0.8U,Selective amplification was carried out using 30×-diluted pre-amplification template.2.Obtained 60 polymorphic bands from 64 primer-combinations60 polymorphic bands have been obtained from different periods of mustard leaf by 64 primer combinations.This 60 polymorphic bands may be related to the red leaf of mustard.3.Cloned five bands from polymorphie bands and compared in NCBI We cloned and sequenced five bands with high repetitiveness and obvious differences from 60 polymorphic bands and compared the sequenced result in NCBI bank.The result show a band with 149bp may belong to chloroplast gene fragment of mustard which may cause the leaf color changed.Other four sequences are similar with Arabidosis thaliana diacylglycerol kinase,Brassica oleraceacy steine protease, Arabidopsis thaliana cysteine-type peptidase,Arabidopsis thaliana peptidyl-prolylcis-transisomerase cyclophilin-type family protein,Arabidopsis thaliana alpha-N-arabinofu-ranosidase/ hydrolase, hydrolyzing O-glycosyl compounds/xylan 1,4-beta-xy homologous respectively.Further research will be done to verify whether these five sequences are related to the red leaf in mustard.