Studies On The AFLP Marker Of The Leaf Color Gene Of Burley Cultivar And Genetic Diversity Of Tobacco Germplasm Resources

Tobacco(Nicotiana spp.) is an important economic crop and has been cultivated by man for thousands of years in many countries.Since its introdution in Ming Dynasty,tobacco has been cultivated in China for more than four hundred years and has produced plentiful germplsms.With the development of tobacco production,more and more germplsms have been introduced into our country in recent years.And now we have held 4042 tobacco accessions. The study of the genetic diversity of tobacco will promote the collection, conservation, identification, innovation and utilization of the tobacco germplasm resource.AFLP is a DNA molecular marker technique with high efficiency, stability and accuracy. It has been widely used in genetic diversity analysis, classification, cultivar identification, genetic mapping, breeding, location of target gene, molecular marker-assisted selection(MAS) in animals and plants. Using this technique,we have studied the marker linked to the the leaf color gene of burley cultivar and genetic diversity of tobacco germplasm.The main results as following:1. The genomic DNA of tobacco was extracted by SDS method and CTAB method respectively and we found that the genomic DNA of tobacco extracted by the CTAB method was good enough for the analysis of AFLP.2. After systematically study on the main factors involved,an AFLP silver-staining analysis system suitable for tobacco genomic DNA was established. The restriction-ligase reaction was accomplished in one step at 37℃for about 6-8h. E+A/M+C primers were used in the pre-amplification reaction.The pre-amplified products were diluted 20 times with 1×TE and used for selective amplification. E+3/M+3 primers were used in the selective amplification and each sample was denatured by adding 7μl Loading buffer and heated up at 95℃for 5 min,then cooled on ice immediately after the selective amplification. The PCR reaction produts are analysed on 6% denatural acrylamide gels and then stained by silver.3.One back cross population was constructed from a burley cultivar L8 and a normally green cultivar Dabaijin599.One marker E-CAA/M-GGC TY260 tightly linked to the leaf color gene of burley cultivar was identified.