Research On Molecular Genetic Mechanism Of Resistance To Heterodera Glycines In Soybean

Soybean cyst nematode（SCN,Heterodera glycines）is a devastating disease in soybean worldwide.The most effective way to manage SCN is combining resistant varities with non-host crop rotation,but crop rotation is restricted by the limited land resources;highly toxic and effective chemical nematicides have been inhibited or restricted in use.SCN is a mixed population with various races in the field.Currently,SCN resistant resources of most soybean varities mainly derived from single-gene resistance,limited resistance sources in northeast China restrict the application of resisant varieties.The main resistant varities appeared lost or weaker in resistance to virulent races.Further,molecular genetic background among these resistant breeding lines was unknown and the molecular resistance mechanism remained unknown which make them not effectively be untilized.Therefore,it is urgently required to screen and breed multiple resistant varieties.Exporing the resistance mechanism can make these resistance breeding lines or cultivars be utilized effectively and speed up the resistance breeding program.The results were as follows:1.Sixty-two soybean genotypes were evaluated for resistance in response to the highly virulent SCN race 4（HG type 1.2.3.5.6.7）and SCN race 5（HG type 2.5.7）.The results demonstrated the great variation in phenotype among these genotypes.Through the specific amplication and sequencing of the rhg1 and Rhg4 loci,three Haplotypes among 51 local genotypes were identified,new resistant type were obtained and the tested main cultivars in Northeast China were all susceptible.The expression level of the rhg1 and Rhg4 loci was associated with copy number accordingly linked to resistance or susceptibility.These identified resistant resources and the linked molecular markers can accelerate marker-assisted selection for SCN resistant breeding program.2.Based on results identified above,one representatively resistant genotype 09-138（Haplotype II）and one local susceptible genotype Suinong54（Haplotype III）were chosen for developing F₂ population through crossing and selfing.The response of all F₂ individuals to SCN race 4 were phenotyped.The wide distribution range of F₂phenotypes indicated multiple gene participation for SCN resistance;extreme phenotypes out of the resistant parent 09-138（FI 9）and the susceptible parent Suinong54（FI 113）in F₂ population showing transgressive inheritance.Through the Graded Pool-seq method,QTLs associated with FI were identified on chromosome 8,10,11,13 and 14 with a total length of 6.5 Mb.Through genotyping by targeted sequenceing（GBTS）with single-marker Kruskal-Wallis analysis（K*）and multiple factor model（MQM）mapping,21 QTLs on 16 chromosomes were identified to contribute 5.7-12.6%phenotypic variation explaned（PVE）to SCN resistance,and both parents contributed SCN resistance,indicating transgressive resistance.The identified common QTL intervals obtained from the two methods（Graded Pool-seq and GBTS）were functionally annotated and some transcriptional factors regulating biotic or abiotic stress were found to be potentially associated with SCN resistance.3.QTL mapping of F₂（Suinong54×09-138）population demonstrated that multiple minor QTLs were involved in resistance and some segragated individuals had lower FI but with tiny root.Therefore,to improve the evaluation index for SCN except FI,for the first time 162 BC₃F₇-BC₇F₃ soybean chromosome segment substitution lines（CSSLs）derived from a local cultivar G.max Suinong14（susceptible recurrent parent,Haplotype III）×wild type G.soja ZYD00006（susceptible donor parent,Haplotype III）were utilized for SCN-CGR resistance evaluation and QTL mapping.Phenotypic results displayed a wide range of distribution and transgressive lines in both FI and CGR for SCN5,and demonstrated a higher correlation between CGR and root weight（R²=0.5424）,compared with that between FI and CGR（R²=0.0018）.Using the single-marker analysis nonparametric mapping test,a total of 38 QTLs（FI and CGR）covering20 soybean chromosomes were identified by two methods,both susceptible parents contributed to transgressive inheritance.The combination of positive alleles for FI,CGR and root weight exhibited low nematode reproduction in the absence of strong resistance genes,such as rhg1 and Rhg4,which will be helpful for effective SCN resistance breeding.Meanwhile,the detected CSSLs tolerant to nematodes are potential replacement in the absence of resistant varieties since they have uniform Suinong14background.4.To analyze the different responses to SCN4 and SCN5 in 09-138,infected and uninfected roots were sequenced with full-length transcriptional sequencing（ONT）.Genome structure analysis obtained 288 lnc RNA transcripts,about 200 alternative splicing（AS）.At least 200 transcripts were found in each transcriptional factor,such as WRKY,AP2/ERF-ERF,NAC and GRAS.Total 2255 differentially expressed genes（DEG）were identified.Through DEG functional annotation and gene-rich analysis,all detected peroxidases were related to nematode defense response（GO:000215）,KEGG was rich in phenylpropanoid biosynthesis（ko00940）;in the clustering analysis of all WRKY transcriptional factors,96%of WRKY-DEG were associated with defense response of respiratory burst（GO:0002679）.Two VQs（Glyma.03G120700 and Glyma.19G125300）had higher expressed level compared with control which might play potential function for nematode defense.To validify these DEG,three time points（3,6 and 8 d）and four soybean varieties（09-138,Kang12,11-452,and Dongsheng1）with different Haplotypes were chosen to perform q RT-PCR.The different expression patterns among two nematodes denote different defense/resistance mechanism.Further functional analysis of these DEG will gain more understanding of SCN resistance/defense mechanism in soybean.In summary,the study including the resistance resources screened,the determined haplotypes,and the identified molecular markers will accelerate marker-assisted selection breeding program for SCN resistance.The analysis of interaction mechanism between soybean and SCN enriches the understanding of plant-nematode interaction.