| With its high protein and oil content,soybean [Glycine max(L.)Merr.],which originated in China,has been a vital part of the human diet.Soybean mosaic virus(SMV)disease is broadly distributed in the world and causes significant yield losses and seed quality deterioration.Presently no chemical drugs can be able to prevent SMV effectively,and promotion and application of resistant soybean varieties is the most economical,effective,and eco-friendly approach for controlling SMV disease.Hence,evaluation of the SMV-resistance levels and screening outstanding resistance resources in soybeans play a vital role both in breeding disease-resistant cultivars and studying the mechanism of resistance inheritance.Additionally,genetic transformation can break the barriers between the species and directionally reconstruct,recombine and transfer the excellent resistance genes,which can greatly shorten the process of breeding disease-resistant cultivars.In particular,the CRISPR/Cas9 genome editing technology develops rapidly and it provides the new strategy for cultivating genetically modified crops resistant to viruses.97 widely-grown soybean cultivars,spanning nine decades(1923-2006)of breeding,from four main soybean-producing sub-regions in China comprising Northern Heilongjiang(NH),Mid-southern Heilongjiang(MSH),Jilin-Liaoning(JL),and Huang-Huai-Hai Rivers Valley(HH),were selected to respectively be inoculated with six prevalent SMV strains including SC3,SC7,SC8,SC11,SC15,and SC18.The average disease index(ADI)of six SMV strains ranged from 26.95 to 48.97.The numbers(percentages)of resistant cultivars and susceptible cultivars for six SMV strains ranged from 27(27.8%)to 64(66.0%)and 33(34.0%)to 70(72.2%),respectively.The ADIs of NH,MSH,JL,and HH were 50.82,47.27,43.10,and 33.05,respectively.ADI values,respectively.In the regions of NH and JL,DI values all exhibited the descendant tendency.In MSH,no descendant tendency of DI values was observed.In HH,DI values for SC3 and SC 18 displayed apparent ascendant tendency,and obvious descendant tendency in DI values for SC15 was observed.Spatio-temporal variation analysis of the SMV-resistance indicates that: NH and JL should maintain and continuously improve the breeding level for SMV resistance;MSH should strengthen the breeding program for all the SMV strains;HH should conduct breeding program especially for SMV strain SC3 and SC18.Twenty-four soybean cultivars were identified to be with the broad-spectrum resistance with the ADI values ranging from 0.80 to 35.52 for the six SMV strains,and totally 13 soybean cultivars were identified highly resistant to at least one SMV strain.The conserved region of SMV HC-Pro gene was confirmed by nucleotide sequence alignment of 13 popular SMV strains.The conserved regions of [ss(+)RNA] and [ss(-)RNA]were at nucleotide sites of 2039-2309 nt and 8448-8467 nt,respectively.Then the PAM loci and the gRNA(+)/gRNA(-)target sequences were determined.CRISPR/Cas9 genome editing vectors were constructed through homologous recombination reactions,and were introduced into the A.tumefaciens strain EHA105 by the freeze-thaw method.24h-germination explants from soybean cultivar Jack(susceptible to SMV)were used for the cotyledonary node-Agrobacterium-mediated soybean transformation system using the bar gene as a selectable marker.319 seedlings were obtained from the tissue culture containinggerminaton,expant preparaton,A.tumefacines suspension preparaton,inoculaton,co-cultiv ation,shoot induction,shoot elongation,rooting,acclimatization,domestication and transplant.83 positive T? plants were obtained through leaf-painting assay(200 mg/1Basta),PCR verification(gRNA,dpCas9 and bar)and LibertyLink? strip detection.For further analysis,796 Ti soybean plants from 31 T? lines were selected,and from which a total of 493 plants were confirmed as transgene-positive.SMV-resistance assessment(strain SC3)? quantitative real-time PCR(qRT-PCR)analysis and serological determination of double antibody sandwich enzyme-linked immunosorbent assays(DAS-ELISA)were employed to detect T i plants from phenotype,RNA and protein level,respectively.SMV resistance was visually evaluated in the Ti generation,and 15 highly resistant(HR;3.0%),53 resistant(R;10.8%),58 mild resistance(MR;11.8%),and 367susceptible(S;74.4%)plants were identified.qRT-PCR analysis was performed at 15 and30 days post-inoculation(dpi)with SMV,the results revealed a gradual reduction and increase in the expression levels of sgRNA!dpCas9 genes and the viral accumulation(SMV CP gene),respectively,as the resistance level of transgenic plants decreased;suggesting that the viral content was negatively correlated with the expression levels of sgRNA!dpCas9genes,and SMV was inhibited by sgRNAidpCas9 gene transcripts which was closely related to the expression levels of the two genes.DAS-ELISA analysis was conducted at 2months post-inoculation with SMV,the results revealed that the values of HR plants were all below 2.0(negative for SMV),and the values increased as the resistance level of transgenic plants decreased which were all above 2.0(positive for SMV).In addition,the phenomenon of seed coat mottling was also investigated in T2 seeds,which showed that the amount of mottled seeds,the mottled scale and the average mottling rates were increased as the resistance level of transgenic plants decreased;in the HR plants,virus-induced seed coat mottling was thoroughly eliminated and none of the T2 seeds were distinguishable from those harvested from the mock-inoculated plants(negative controls);the mottled scale on the seeds differed obviously between the positive controls,R,MR and S plants,however,there was no big difference in the average mottling rates of them,indicating that the seed coat mottling was induced in most of the progeny seeds by a small quantity of SMV virus,and the mottled scale was negatively correlated with the resistance level of transgenic plants.CRISPR/Cas9 genome editing technology provides a new strategy for breeding soybean cultivars with broad-spectrum and persistent SMV-resistance. |
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