Analysis Of Gene Expression In Wheat Roots Infected By Heterodera Filipjevi And The Resistance Identification Of Wheat Germplasm Resources

Heterodera filipjevi is a great threat to globle cereal production.In China,the outbreak of H.filipjevi is mainly take place in Henan province,and causes substantial losses in wheat production.In recent years,H.filipjevi parasitism become more and more serve along with the development of agricultural intensification and mechanization,and it is necessary to reinforce the study on the mechanism of this pest as well as the resistance identification of a variety of wheat cultivars so as to enhance the control techniques.So far,little is known about the molecular mechanism concerning the H.filipjevi parasitism,and no study on the microassay analasis of gene expression response to the infection of cereal cyst nematode(CCN),as well as characterization about the resistance of a great variety of wheat cultivars or close species has been found.Otherwise,it has been approved that the utilization of high-resistant varieties is the most effective and economical strategy to the control of CCN pest.Unfortunately,most cultivars widely used in present wheat production in China have been proved to be susceptible to CCN,thus the exploration of resistance genes from the close germplasm occur to be most importance.little reports on this respects have been documented.Wheat cultivar Chinese-spring,a cultivar beyond those widely-used and well-known for its weakness in disease resistance and adaptability,is suitable to the study of wheat genetics as well as molecular pathology,while several wheat relative genera species have been found to carry abundant resistance genes and desirable traits,and some of them may contributed a lot to the resistance of wheat to H.filipjevi.Basing on that,in the present study,we carried out following studies:(1)We used the customized Agilent 4×44K wheat whole-genome oligo microarray containing 43803 probe sets and covering over 42000 transcripts to investigate early gene expression profiles in wheat roots infected with H.filipjevi Xuchang population.Statistical analysis indicated that 820 genes monitored were identified to be differentially expressed genes after Heterodera filipjevi infection,of which 496 transcripts were up-regulated and 324 transcriptswere down-regulated.Of total 820 differentially expressed genes,317 genes had been annotated.The Gene Ontology analysis revealed that accounts for 17.35% of these genes were involved in amino acid/protein metabolism,energy metabolism,carbohydrate metabolism,fatty acid and lipid metabolism,23.97% involved in 18 transcription families,such as AP2-EREBP,b ZIP,C2H2,MYB,TLP,WRKY,etc,15.14% involved in signal transduction(synaptic signaling,ras-related protein,signal-transducing G protein,etc),3.15% involved in cell structure,5.36% involved in stress/defence,such as NBS-LRR disease resistance proteins,8.52% involved in transport,12.30% involved in protein synthesis,and other genes were unclassified.We focused on functional categories related to cell structure,stress/defence,transcription factors,and signal transduction.(2)A total of three differential WRKY genes from transcription factors were randomly chosen for analysis time course expression of susceptible wheat using real time quantitative reverse transcription PCR(q RT-PCR).The results indicated that WRKY and WRKY74-a genes were differenttly expressed in susceptible varieties Chinese Spring and Xinmai 18,at 48 hpi and 168 hpi,the WRKY gene was shown significant difference in Chinese Spring and Xinmai 18.otherwise,there are shown no significant difference at other point in time.The WRKY74-a gene was similar to WRKY.The WRKY8 gene was highly expressed in both Chinese Spring and Xinmai 18 at early infection stage.However,the expression of WRKY8 gene decreased gradually in both Chinese Spring and Xinmai 18 with the extension of inoculation time.(3)To mine resistance genes and resistant resources from wheat relative species and germplasm resources,we identified the resistance to H.filipjevi in wheat relative species and 26 germplasm resources by inoculating H.filipjevi Xuchang population with relative resistance index method in greenhouse,and we also identified the resistance to H.filipjevi Xuchang population in 26 germplasm resources through field test.The results showed:(1)One candidate(PI542187)of 34 accessions of Aegilops geniculata were identified with high resistance and 15 candidates with resistance to H.filipjevi Xuchang population,proving that there are a great number of resistant resources in Ae.geniculata.Three Chinese Spring-Ae.ovata chromosome addition lines(DA 3Ug?MA 7Ug?MA 5Mg)were identified with resistance to H.filipjevi Xuchang population.We identified the resistance to H.filipjevi in Chinese Spring-Ae.ovata chromosome translocation lines 5Mg-5D,four chromosome translocation lines(5Mg464?5Mg466?5Mg511and 5Mg494)were identified with resistance,and T550 which is the 5Mg chromosome carrier was identified with resistance.Otherwise,Chinese Spring was identified with moderate susceptibility.Three Chinese Spring-Ae.ovatachromosome additional lines(DA3Sv?DA4Uv ? DA6Uv)were identified with resistance to H.filipjevi.All of this leads to the conclusion that the three chromosomes(3SV?6UV and 4UV)of Ae.ovata carry the resistant genes to H.filipjevi.The Chinese Sprting-Ae.speltoides chromosome additional lines DA7 S was identified with high resistance to H.filipjevi.Proving that the 7S chromosome of Ae.speltoides have the resistant genes.two Chinese Spring-Ae.caudata chromosome additional lines(DAC#1and DAE#1)were identified with high resistance.(2)Two Chinese Spring-Ag.elongatum chromosome additional lines were identified with resistance to H.filipjevi and other four candidates were susceptible.(3)There are one candidats(HN15)with resistance to H.filipjevi in 18 Chinese Spring-D.villosum chromosome translocation lines,and five candidates(6VL?HN9?HN10?HN11and HN14)were identified with resistance.(4)In 26 germplasm resources,only five candidates(XD11-2,XD11-8,XD11-21,XD11-25 and XD11-34)were identified with resistance to H.filipjevi in greenhouse and field tests.The identified results of six candidates in greenhouse were inconsistent the results in field tests.In future,we can directly use the XD11-8(Longmai 30)as a good germplasm resource for wheat breeding with the good resistance to H.filipjevi.As our knowlege,this is the first report on the analysis of gene expression in wheat roots response to H.filipjevi using the recently developed wheat microarray.Our study revealed the a series of genes diffrentialy expressed response to the infection of H.filipjevi,and these genes mainly concerned to the reconstructure of cell structure,defense and strsss,translation regulation,and signal transduction.Some of these diffrentially expressed genes were further proved by the real time q RT-PCR assays,supporting the results of reliablity of the microarrays.In addtion,We provided first-hand experimental results on the resistance data of a series of wheat relative genera species to H.filipjevi.Our study reveal the molecular strategy concerning the infection of H.filipjevi,may serve a useful reference to the further study on the interaction of nematode-host,selection of resistance gene as well as resistant-variety breeding.