Relationship Of Pod Shattering Resistance With SHAT1-5 Gene Structure And Expression In Soybean

The pod shattering phenomenon is one of the key factors contributing to the loss of yield in wild soybean（Glycine soja）.At present,the expression level of SHAT1-5,which is identified by Dong et al.,equal to 15 times of the wild soybean allele in cultivated soybean pods,therefore formed the pod resistance in cultivated soybean.Further studies have shown that the high expression of SHAT1-5 gene in cultivated soybean is due to the absence of a 20 bp deletion located at 4 kb upstream of the gene.The preliminary study in our laboratory found that the 20bp deletion in the cultivated soybeans accounted for only 14.6%,and the Fst between the wild soybean and cultivated soybean is also small,then how to explain the pod shattering phenomenon in other 85.6%cultivated soybean which does not contain the site?How does their relationship with the SHAT1-5 gene,which need to be further studied.In this study,75 representative soybean（48 cultivated soybean and 27 wild soybeans included）were selected act as study materials,in order to seek the functional polymorphism sites associated with the resistance to pods,combined with bioinformatics analysis the genetic structure and expression differences of SHAT1-5gene between soybean wild population and cultivated population,and to provide a theoretical basis for further analysis of the relationship between these loci and pod shattering in soybean.The main results of this study are as follows:1.The SHAT1-5 gene sequence was found in the Phytozome database.The bioinformatics analysis revealed that the gene was simple and contained three exons and two introns.SHAT1-5 encodes a protein containing 443 amino acids.There is a NAM subdomain in the amino acid sequence by amino acid sequence blast.The physicochemical properties and structure of the protein are predicted.The results show that there is no transmembrane domain in the protein and located at the cytoplasm,three domains existed in the secondary structure,including chain structure,helix structure and spiral structure.The three-dimensional spatial structure predicts that the gene is a stress-induced NAC1 transcription factor.2.A 7098bp DNA consensus sequence was obtained by sequencing the SHAT1-5genome and the promoter sequence of 75 soybean materials（including 48 cultivated soybean and 27 wild soybeans）.A total of 70 nucleotide polymorphisms were found,including 54 single base substitutions and 16 indels.The genetic diversity of wild soybean and cultivated soybean were 1.448×10^-3and 0.438×10^-3,and the genetic diversity was reduced 69.8%during domestication.The genetic differentiation coefficients of these 70 polymorphic loci between soybean wild populations and cultivated populations were analyzed.Based on the distribution of wild soybean sub-allele frequencies in cultivated soybean,3 anti-pods-related functional sites were found,which are located in the promoter sequence,and no functional polymorphism sites associated with anti-pod were found in the genomic region of the SHAT1-5 gene.3.The temporal and spatial expression characteristics of SHAT1-5 gene in wild soybean and cultivated soybean were analyzed by fluorescence quantitative PCR using cultivated soybean 261M and wild soybean 041S as experimental materials.The results showed that SHAT1-5 gene was mainly expressed in pods（18 days after flowering）and stem in the spatil,the expression of SHAT1-5 was the highest in pods（18 days after flowering）from the temporal.Whether in space or in time,the expression pattern of wild soybean and cultivated soybean was similar,and the expression level of cultivated soybean was almost higher than that of wild soybean.Considering of the expression and gene structure of SHAT1-5 gene in wild soybean and cultivated soybean,and three functional sites related to the resistance of the gene were further screened.In this study,the differences of genetic structure and expression of SHAT1-5gene in wild soybean and cultivated soybean were analyzed by sequencing technique and fluorescence quantitative PCR technique.Three functional polymorphic loci related to high expression of SHAT1-5 gene were initially screened out.Which can provide a theoretical basis for further analysis of the relationship between these loci and pod shattering resistance in soybean.