A Study Of The Isolation, Identification And Inter-Simple Sequence Repeat (ISSR) Analysis Of Fusarium Oxysporum F.SP. Lini On Linseed(Linum Usitatissimum L.)

Linseed(Linum usitatissimum L.), a member of pedaliaceae family, is one of the most important economic crops. The incidence rate of Fusarium wilt is about10%-30%, even more than50%in recent years. Thus Fusarium wilt has significant impact on linseed yield and quality. Fusarium wilt was caused by Fusarium oxysporum f. sp. lini which can infect linseed in whatever growth period. The study of isolation and identification of Fusarium oxysporum f. sp. lini, genetic diversity analysis and resistance breeding has just started compare to the other crops such as melons etc.The696Fusarium fungi were isolate from the infected plants from six provinces that had got in this research. Through morphological identification, we got typical isolates sequenced their internal transcribed spacer (ITS) regions of the ribosomal RNA gene, which allowed the identification of isolates at the species level. Finally we got96Fusarium oxysporum f. sp. lini isolates for ISSR analysis. The main results are as follows:1. An Orthogonal design L16(45) is applied to the optimization of ISSR-PCR reaction system for Fusarium oxysporum f. sp. lini. A most suitable ISSR-PCR reaction system was established. The optimized reaction system consists of20ng template DNA,1U Taq DNA polymerase,0.6μmol·L-1primer,0.15mmol·L-1dNTPs,3.0mmol·L-1Mg2+and2.5μL10×buffer, in total25μL volume. Optimal annealing temperature was decided by different primers.12primers with stable amplification and rich polymorphism for ISSR were selected.2. Twelve ISSR random primers amplified a total of722DNA loci, percentage of polymorphic loci is99.45%. Each primer was amplified60.17bands. The size of all products was between200-5000bp. The genetic identity varied from0.9579to0.9803, the average value is0.9688.3. The genetic diversity of individuals was critical. Genetic variation between populations accounted for81.02%of the genetic variation within populations. The most of the genetic variation derived from the population between genetic variations. There was a certain gene flow between the Fusarium populations.4. The six populations were clustered into three subgroups.96Fusarium isolates were divided into five groups when the similarity coefficient was0.870. There was a relationship between the Fusarium oxysporum ISSR groups and their geographical origin. The96Fusarium oxysporum f. sp. lini had higher genetic similarity, larger genetic differentiation and a wealth genetic variation.