Evaluation And Application Of Near-isogenic Lines For Allergenic α-subunit Deficiency Of 7S Globulin In Soybean(Glycine Max L.Merrill)

α-subunit of 7S globulin(β-conglycinin)is one of the major components of storage protein in soybean and was identified as major allergen.The presence or absent of α-subunit significantly inflences soybean end-products quality,therefor,understanding the effects of α-null trait on soybean protein quality will be very useful for soybean protein quality improvement.In the present study,through a genome-wide DNA marker screen combined with stringent phenotypic selection,we developed near-isogenic lines(NILs)of soybean with the cgy-2 allele for allergen-α-subunit-deficiency of β-conglycinin on chromosome 20 derived from a cross between the Chinese high oil cultivar soybean ‘Dongnong 47’(DN47)and the α-null allele donor parent‘HS99B’,and Named as‘cgy-2-NIL’.The developed NILs represent particularly powerful materials to isolate and characterize the cgy-2(α-null)genes and will be useful to study the genes responsible for its unique biology.Using‘cgy-2-NIL’ and its recurrent parent ‘DN47’,we evaluated the effect of allergen-?-subunit-deficiency on soybean amino acids(AAs)composition and To-fu processing quality.Furthermore,to identify α-null-related transcriptional changes,the gene expressions of cgy-2-NIL and its recurrent parent DN47 were compared using Illumina high-throughput RNA-sequencing of samples at 25,35,50,and 55 days after flowering(DAF).Seeds at 18 DAF served as the control.The main results are as follows:(1)After 4 generation crossing and backcrossing assisted by molecular-marker assisted background selection,combining with stringent phenotypic selection,we developed near-isogenic lines(NILs)of soybean with the cgy-2 allele,and named it as‘cgy-2-NIL’.The substituted region of the NILs was narrowed down to a physical distance of approximately 11.18 c M only on chromosome 20.The agricultural characteristics of most of the developed NILs were very similar to those of ‘DN47’.(2)Near-isogenic line‘cgy-2-NIL’ and its recurrent parent ‘DN47’ was used to investigate amino acid content and nutritional quality.Crude protein content,total AA content,total essentialamino acid(EAA)content,and sulfur-containing(Met and Cys)amino acids increased by 4.11%,4.16%,5.20%,and 11.96% in‘cgy-2-NIL’ compared with ‘DN47’,respectively.(3)There is a general increase in the constituent level of most amino acids and free amino acids content in‘cgy-2-NIL’.The increased synthesis of free Arg makes a drastic contribution to the high free amino acids of ‘cgy-2-NIL’ mutants.The cgy-2 allele is an alteration of a functional allele that is closely related to soybean protein amino acid quality,and is useful for hypoallergenic soybean breeding programs that aim to improve seed protein quality.(4)Analyses of total essential amino acid(TEAA)and the EAA index(EAAI)revealed that TEAA and EAAI of ‘cgy-2-NIL’were higher than in ‘DN47’.The null-allele of the ?-subunit positively affects the AA scores,so α-null mutant can remove undisirable α-allergen subunit and simultaneously improve the seed nutritional value.(5)By RNA-Seq,a total 20295 differentially expressed unigenes were detected which could be subdivided into 174,151,123,158,and 2837 genes that varied in abundance at 18,25,35,50,and 55 DAF,respectively.The largest numbers of DEGs were identified at 55 DAF.The majority of DEGs showed development-stage-specific expression.Seventeen DEGs were differentially expressed in all five stages.The top 20 genes that showed high-level differential expression related to the α-null mutation.We hypothesized that these genes are putatively ?-null-related transcripts.Based on obtained DEGs information and bioinformatics,we will conduct further studies focused on gene function identification of the above-mentioned DEGs.(6)GO category enrichment analysis(P-value < 0.05)revealed different results in different stages.A similar GO category distribution pattern of transcripts was found at 18,35,and 55 DAF.Howevre,the DEGs identified at 25 DAF represented a unique pattern of GO category distributions.KEGG pathway analyses identified 541 altered metabolic pathways in cgy-2-NIL.At18 DAF,12 DEGs were involved in arginine and proline metabolism.(7)In the present study,74 TFs were differentially expressed between cgy-2-NIL and DN47,when a fold change ≥ 1 and P < 0.05 were used as cutoff values.These genes were divided into different classes.These TFs included BREVIS RADIX,GRAS,jumonji,GATA,SBP-box,and TCP.The most abundant TF group was GRAS.Notably,13 MADS-box TFs were all downregulated at55 DAF.(8)The cgy-2 allele in the homozygous form modified the expression of several Cupin allergen genes.Notably,the α-null mutation was associated with significantly reduced expression of both α-subunit genes,Glyma20G28650 and Glyma20G28660,in proportional amounts.In addition,the expressions of Gy1,Gy2,Gy4,and Gy5 in cgy-2-NIL were all lower than that in DN47 throughout five developing stages,and these genes showed a similar pattern of expression,(9)We used q RT-PCR to validate selected DEGs identified from the RNA-seq data.All five selected genes showed basically consistent up or downregulated expression patterns at 35 and 55 DAF,respectively,confirming the RNA-seq data.