| Late blight,caused by oomycete pathogen Phytophthora infestans is the most destructive disease in potato production worldwide,results in 15% loss of the potato production per year.To prevent and minimize potato disease loss,chemical fungicides were extensively utilized,which increases economic investment and leads to serious environment pollution.The most economical and effective measure for preventing late blight is to breed varieties with broad-spectrum and durable resistance.However the lacking of resistance resources and the complexity of genetic background severely restricted the efficiency of disease resistance potato breedingin China.Therefore,the breeding for stacking R genes with NBS-LRR domain is the most effective breeding method in potato in the world.Base on previous collected late blight resistance resources and study,this work aims to explore known R genes or identify novel disease resistance genes in excellent resistant materials by screening a number of potato late blight resistance materials and using the pathogen inoculation,R gene molecular markers,effectoromics strategies and d Ren Seq technology.This study will provide a resource basis for assisting in stacking of R genes,breeding broad-spectrum and durable potato varieties,and original materials and technical support for the cloning of novel R genes.The main results are as follows:1.240 potato materials were inoculated with three P.infestans strains 16-2,3928 A and 14-2 to evaluate late blight resistance,and 36 middle to high level resistant potato materials were determined.In further investigation,the 36 potato materials were infected with three P.infestans isolates GN51,HTP-9 and NL11564,which are derived from Yunnan,Wuhan and Netherlands respectively,to test their resistance spectrum.Finally,we found that most of the materials maintain resistance to GN51;the novel HTP-9 strain can overcome 14% resistant materials,and 50% of resistant material can be infected by NL11564.2.PCR assay demonstrated that R8 gene can be amplified in 20 resistance potato materials,such as 'Jiaxiang No.1',indicating that R8 gene exists in resistance materials with high frequency.Transient expression analysis showed that P.infestans effector Avr8 cloned from seven different P.infestans races can be recongnised by R8.It is speculated that this is the main reason that R8 carrys broad-spectrum resistance to P.infestans.But,we found most of R8 resistance materials could be infected by the pathogen race NL11564.3.The d Ren Seq was performed to analyze R genes seqences in 24 representative potato resistant materials and 8 known R genes were found in materials,including 6 P.infestans resistance genes(Rpi)R8,R3 a,R3b,R1,Rpi-blb1 and Rpi-ptal,a virus resistance gene Rx,and a nematode resistance related gene Gpa2.Among these materials,there are 6 Rpi genes were found in one high-generation breeding parent 06HE13-1.4.To confirm d Ren Seq results,R1 and R8 genes were amplified from the resistant material 06HE13-1 according to the diagnostic results.There are two types of R1 and R8 were cloned,and the R1 and R8 with correct length can be recogniaed by Avr1 and Avr8 respectively.The result demonstrats the accuracy of the d Renseq in the study.5.38 P.infestans effectors were transiently expressed in leaves of resistance materials by PVX-agroinfection technique.50% effectors infected sites showed necrotic spots in 12 resistance materials and a few necrotic spots were observed in 16 materials,suggesting that there might be some unknown R genes in these resistant materials.But the conclusion need more biological repeats to verify.6.The ploidy identification of 36 resistant materials showed that these materials contained 6 diploid,21 tetraploid,1 pentaploid,and 4 hexaploid.Agronomic traits survey showed that most of material shown good and diverse agronomic traits. |
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