Establishment Of Duplex-pcr Detection System And Development Of Microsatellite Markers For Heterodera Avenae

Cereal cyst nematode(CCN)Heterodera avenae and H.filipjevi are the main pests on cereal crops and cause great losses to yield in China.H.avenae has been confirmed to distribute and occur in 16 provinces,while H.filipjevi has been found in Henan and Anhui Provinces.The co-infection of H.avenae and H.filipjevi was discovered quiet common in wheat field of Henan Province.In this study,the duplex PCR detection system for detecting H.avenae and H.filipjevi simultaneously was established in order to rapidly diagnosing the complex infection situation of CCN in wheat fields and benefit to the integrate management strategies.The microsatellite markers(SSR)of H.avenae were developed using the EST database of in GenBank which can be further used for analyzing the genetic structure and diversity of Chinese populations of H.avenae,and elucidating the possible domestic introduction routes of H.avenae.The main results were as follow:1.Establishment of duplex-PCR detection for H.avenae and H.fllipjeviThe forward primers HaF8 and HfF9 respectively specific to H.avenae and H.filipjevi together with the common reverse primer HafR8 were designed according to the comparisons of mtDNA-COI sequences from 24 populations of 10 PPN species.The single PCR product for H.avenae amplified with primers HaF8/HafR8 was 200 bp,for H.filipjevi with primers HfF9/HafR8 was 320 bp.The band sizes were easily been distinguished.A high efficient duplex-PCR detection system was developed for these two CCN species with the optimized concentration of HaF8/HfF9/HafR8 as 0.24:0.16:0.4 ?mol/L at the annealing temperature 58?,which could identify H.avenae and H.filipjevi simultaneously from all detected samples.The sensitivities of the duplex-PCR detection for single cyst of H.avenae and H.filipjevi were both 1/2000000,but for single second stage juvenile were 1/640 and 1/1280,respectively.The species of CCN samples collected from wheat fields in Huanghuai region of China were successfully detected by the duplex-PCR,which demonstrated the possibility for application in rapid diagnosis of the complex infection situation of CCN in wheat fields.2.Development of SSR markers for H.avenae based on the EST DatabaseUsing the EST database in GenBank to develop microsatellite markers(SSR)is the most economical and efficient strategy.In this study,210 pairs of SSR primers were designed based on the EST sequences of H.avenae,and 13 pairs of primers with high polymorphism were obtained by screening of touch-down PCR and typing of short tandem repeats(STR).Polymorphism analysis of the 13 pairs SSR primers was preformed on population JSPX of H.avenae,and the result showed that 8 of them,including CS-31,CS-60,CS-102,CS-121,CS-137,CS-179,CS-187 and CS-8384,have the null allele frequency with low values,even up to 0,as well as the PIC(polymorphism information content)with values larger than 0.25,which indicating their high polymorphism.The Hardy-Weinberg balance test and Linkage genetic test were carried out for 13 screened SSR and the P values were corrected with Sequential Bonferroni.The above 8 SSR markers neither deviated Hardy-Weinberg balance(P<0.05),nor existed the Linkage genetic phenomenon(P<0.05).These results indicated that 8 SSR markers were confirmed with high polymorphism which can be further used for analyzing the genetic structure and diversity of geographical populations of H.avenae in China.