Genetic Analysis Of 25 Sichuan Sun-cured Tobacco Germplasm By Sequence Related Amplified Polymorphism (SRAP)

Sun-cured tobacco was not only the important production materials of cigar and mixed type tobacco,but also the important germplasm resources for proceeding cross breeding of Chinese tobacco.There had a long history for planting and cultivating sun-cured tobacco and abundant germplasm resources in landraces of sun-cured tobacco in Sichuan Province.However,nowadays the working of introduction,propagation and keeping seeds that was lack of systematic management was accomplished by tobacco farmers.Besides,due to lacking standard denomination method for tobacco,tobacco was named by tobacco farmers on the basis of the shape of stock and the feature of leaf blade.It was easy to cause confusion phenomenon of homogeneity synonym or heterogeneous isonym,when the method was apply to name the un-clear morphologic characteristics of tobacco.And because of lacking long-term systematical research and management,it led to the un-clear variety sources of sun-cured tobacco and the un-distinct genetic relationship among varieties from many places in Sichuan Province,which had become the limited factors for improving and utilizing sun-cured tobacco.Identifying the germplasm resources of sun-cured tobacco was the basis of quickly improving and utilizing sun-cured tobacco.Molecular mark technique, especially SRAP mark technique that was applied to study on germplasm resources of sun-cured tobacco had few reports in domestic or abroad.So,SRAP mark technique applied to study on genetic diversity of germplasm resources of tobacco,especially research analysis and identification evaluation sun-cured landraces tobacco,was helpful to more exactly identifying and evaluating existent germplasm resources,and providing more dependable reference and election method for reasonable utilization of germplasm resources in landraces of sun-cured tobacco and incubation of good new varieties.In the paper,comparing with 1 flue-cured tobacco and 1 yellow flower tobacco, genetic relationship among 25 sun-cured landraces tobacco materials from Sichuan Province were analyzed by SRAP mark technique.Parts of specific mark straps were recovered and sequenced and analyzed by using bioinformatics analysis.And the following results are received:1.An optimized SRAP-PCR reaction system of Sun-cured tobacco was established.In this study,optimum concentrations of each components in 25μL reaction system were as follows:1.6 mmol/L Mg²⁺,1×Buffer,0.2 mmol/L dNTP,2 ng/μL template DNA,1 U Taq DNA polymerase,0.5pmol each primer.The PCR reaction program was.predenature at 94℃for 5 min,then 5 cycles at 94℃for 30 s, 33℃for 60 s,72℃for 90 s;35 cycles at 94℃for 60 s,53℃for 60 s,and 72℃for 1 min,and finally extended at 72℃for 8 min,keep the PCR products at 4℃.After optimization,the SRAP-PCR reaction system had higher sensitivity,stability and reproducibility.2.Among the 30 pairs of SRAP primer combinations,25 were the found polymorphic and used to analyze 27 tobacco materials.3368 amplification bands were detected.Of which,998 fragments were polymorphic,and the average percentage of polymorphic bands was 29.6.The results showed that this technique is highly efficient for identifying the germplasm resources in landraces tobacco materials from Sichuan Province.3.The similarity coefficient of genetic among 27 materials ranged from 0.26 to 0.97,average similarity was 0.62;Genetic difference of Nicotiana rustia was bviously different to 26 common cultivar tobacco,when coefficients of genetic resemblance were between 0.26 and 0.51;coefficient of genetic resemblance between flue-cured tobacco and 25 sun-cured tobacco was more lower,as coefficients were between 0.66 and 0.78;coefficients of genetic resemblance of 25 sun-cured tobacco were between 0.75 and 0.97,which average value was 0.86,and genetic similarity of 25 sun-cured tobacco was more higher,genetic similarity of sun-cured tobacco in landraces existed different,but the total genetic basis was relatively narrow.4.Coefficients of genetic resemblance of 27 materials were analyzed by UPGMA.The result of cluster analysis indicated,yellow flower tobacco,flue-cured tobacco and sun-cured tobacco could be separated and its genetic differences were reflected,when coefficient of genetic resemblance was 0.74.25 sun-cured tobacco were classified into 3 big groups and 2 independent groups;mao tobacco and liu tobacco belonged to 2 traditional groups of sun-cured tobacco in Sichuan were not obviously clustered into 2 groups,when coefficient of genetic resemblance was 0.89.5.Parts of specific straps marked by SRAP were recovered and sequenced,and parts of sequencing results were preliminarily analyzed by using bioinformatics.It demonstrated that effect of recovering was much better,the length of recovering fragments were about 200bp,and a large of fragments except EU430295 could found their higher homology sequences by proceeding Blastn and BlastX comparing,in order to suppose their protein function.