Identification Of The HS1-2 Resistant Genes In Beta Vulgaris And Functional Analysis Of Lethal Genes From Heterodera Avenae By RNAi

Sugar beet?Beta vulgaris L.?is an important crop providing about one-quarter of world sugar production.The beet cyst nematode?BCN?is a severe pest for sugar beet causing up to 25-50%yield losses.Resistance breeding has been a major goal for nematode control.An effective nematode resistance source was found in Patellifolia procumbens.The related resistant translocation lines TR363and TR520 harbor translocation part from chromosome 1 of P.procumbens.Only TR520 contains the cloned BCN resistance gene Hs1^pro1.There is a strong suggestion of a second gene on both translocation lines and named Hs1-2.Gamma irradiation of TR520 resulted in the identification of two new susceptible translocation lines TR320 and TR659,which lost part of the alien translocation part from P.procumbens.The aim of our research was to exploit whole genomic sequencing?WGS?of TR520,TR363 and TR659 to indentify the second nematode resistant locus Hs1-2.In total,1,822,146,316 reads were obtained from WGS.The reads belong to sugar beet and susceptible translocation line TR659 were successfully removed by mapping and collected the unmapped reads.Finally the full length wild beet specific sequences of Hs1-2 locus were confirmed to 375kbp.In total,10 ORFs were selected in this region for further analysis.RT-qPCR was performed to measure the expression profiles of the ORFs in response to nematode infection in TR520 at 3,7,15 and 25 dpi.The transcript levels of ORF901,ORF903,ORF906 and ORF910 were significantly down regulated after nematode infection at 7dpi and 15dpi;ORF902,which is homologous to Calcineurin B-like gene?CBL?,was up regulated at 7dpi;ORF904 which is highly homologous to a MAP3K delta-1 protein kinase was up regulated soon after nematode infection?3dpi?.Those data suggest that ORF902 and ORF904 may pacitipated in the early stay for nematode resistance and are interested candidate genes of Hs1-2.ORF905 was predicted to encodes a basic-leucine zipper transcription factor like gene?bZIP?.ORF908 was predicted to encode a F-box protein.After nematode inoculation,ORF908 was significantly up regulated at all time points tested and ORF905 was significantly up regulated at 3dpi,7dpi,15dpi.Those data suggest that the ORF905 and ORF908 are interested candidate genes of Hs1-2.After down regulating of ORF902 or ORF904 by RNAi in hairy root,the resistant phenotype has not been changed which indicates that those two genes are not invoved in Hs1-2 locus mediated BCN resistance.Finally,ORF905 and ORF908 were selected as candidate genes in Hs1-2 locus.We analyzed lethal candidates genes and far genes from Heterodera avenae by in vitro RNAi.The results showed that after treated with dsRNA of 2 far genes and 2 lethal candidate genes,the infection rate were decreased when compared with treated with dsRNA of eGFP.