Cloning Of A Gene For Seed Shattering And Identification Of QTLs For Seed Dormancy In Sorghum B140

Domestication is an important process of wild plants to crops,and loss of seed shattering is an important achievement in the human civilization history.Seed shattering is conducive to the normal reproduction of crop species,but the difficulty-to-shatter trait in crop relatives is easy for human harvest,and can avoid severe reduction in yield.So seed shattering is strongly selected in the process of domestication.Seed dormancy is regarded as the phenomenon in which the vigorous seeds are unable to germinate under the suitable germination condition,which is another important process during the domestication of major crops.In this study,map-based cloning and function analysis of shattering gene from weedy type of sorghum variety B140,which showed strong seed shattering when seed mature in the natural condition was conducted,and the seed dormancy related QTL analysis were also done.The main results were as follows:1,through genetic analysis and fine mapping using the F2,F3 populations derived from crossing of B140(a shattering weedy type)with CK60B and MS138B(non shattering type),a candidate gene of sorghum seed shattering,Sh-B140 was narrow-downed into a 5.8 kb region in chromosome 1 between markers SB12970-4 and N24,and in this region,only one gene Sb01g012970 was found.Protein structure prediction showed that Sb01g012970 contains three structural domains:WRKY DNA Banding Domain,DNA Transposase Domain and Zinc Finger Domain,which might be a DNA binding transcription regulator.2,to verify whether seed shattering phenotype was controlled by Sh-B140,we generated a genome vector and transformed into a Sudangrass variety Hiro-1 and got the T1 generation of transgenic plants.Field observation showed that the seed shattering in T1 plants were higher than Hiro-1,but significantly weak than B140.The RNAi victors were undergoing.3,Sh-B140 has high expression level in the abscission layer but less expressed in other tissues;compared with other non-shattering materials such as CK60B,MS138B and Hiro-1,the expression level was significantly decreased at the late booting stage.The Sh-B140-GFP fusion protein(p35S::Sh-B140-GFP)was only localized to nuclei,agreed with its character of transcription factor protein.The results of Yeast two-hybrid system indicated that the gene Sh-B140 was not interacted with both shattering genes Sh1 from wild sorghum and SpWRKY from S.propinquum,a closely relative of cultivated sorghum.4,the expression levels of Sh-B140 showed clear difference between B140 and Hiro-1 when sorghum seedling treated with ethylene after 8 hours.In addition,the expression level of Sb04g023810,a homologues in sorghum of the Arabidopsis SERK1 gene,which negative regulating organ separation in Arabidopsis flowers was analyzed in B140,CK60B,MS138B and Hiro-1,the results showed that the gene Sb04g023810 had a similar expression pattern with Sh-B140 in the abscission layer at booting stage,and the expression level of Sb04g023810 was clearly upped in the T0 plants of over-expression of Sh-B140,indicating some relationship between genes SERK1 and Sh-B140.5,sequence alignments of Sb01g012970 in 12 sorghum varieties(including 2500 bp before the start codon ATG)showed that four bases in the promoter region are lack in all shattering varieties.By sequencing of two cloned sorghum shattering genes,Sh1 and SpWRKY in these sorghum varieties,some inconsistent were found on the domesticated loci of the Sh1 and SpWRKY.We guess different domestication patterns of shattering gene may be contributed to different varieties in sorghum,and the Sh-B140 gene from weedy type of sorghum may be occurred before selection of seed shattering in the process of wild sorghum domestication.6,to identify the genomic regions controlling the seed dormancy in sorghum,we used 118 F2 segregating populations derived from crosses between a weedy type of sorghum B140(deep dormancy-type)as a female parent and sorghum breeding materials,CK60B(week-dormancy-type)as male parents,respectively.Two QTLs for seed dormancy were detected,one QTL was located between markers Xtxp327 and Sam49411b on chromosome 4 and explained 17.9%of the phenotypic variance,and the other QTL was detected between markers Sam73522 and SB4087 on chromosome 7 with 27.8%phenotypic variance explained.Two QTLs for seed dormancy detected will be used for further fine mapping,cloning candidate genes and has laid a solid foundation for cultivating suitable dormancy sorghum varieties.