Applying genotyping (TILLING) and phenotyping analyses to elucidate gene function in a chemically induced sorghum mutant population

Authors: Xin, Zhanguo; Wang, Ming; Anglin, Noelle; Burow, Gloria; Franks, Cleve; Pederson, Gary; Burke, John

Submitted to: BMC Plant Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/14/2008
Publication Date: 10/14/2008
Citation: Xin, Z., Wang, M.L., Barkley, N.L., Burow, G., Franks, C., Pederson, G., Burke, J. 2008. Applying genotyping (TILLING) and phenotyping analyses to elucidate gene function in a chemically induced sorghum mutant population. Biomed Central (BMC) Plant Biology. 8:article 103.

Interpretive Summary: A sorghum mutant population consisting of 1,600 lines was generated from the inbred line BTx623 by treatment with the chemical agent ethyl methanesulfonate (EMS). Numerous phenotypes with altered morphological and agronomic traits were observed from M2 and M3 lines in the field. A subset of 768 mutant lines was analyzed by TILLING (Targeting Induced Local Lesions IN Genome) using four target genes. A total of five mutations were identified resulting in a calculated mutation density of 1/526 kb. Two of the mutations identified by TILLING were verified by sequencing in the gene encoding caffeic acid O-methyltransferase (COMT) in two independent mutant lines. The two mutant COMT lines segregate for the expected brown midrib (bmr) phenotype, a trait associated with reduced lignin content and increased digestibility. This report demonstrates that TILLING as a reverse genetic approach can be successfully applied to sorghum. The diversity of the mutant phenotypes observed in the field, and the density of induced mutations calculated from TILLING, indicate that this mutant population represents a useful genetic resource for members of the sorghum research community. Moreover, TILLING has been demonstrated to be applicable for sorghum functional genomics by evaluating a small subset of the EMS-induced mutant lines. This mutant population will play important role in defining the function of genes identified from the sorghum genome sequencing project.

Technical Abstract: Sorghum (Sorghum bicolor L. Moench) is ranked as the fifth most important grain crop and serves as a major food staple and fodder resource for much of the world, especially in arid and semi-arid regions. The recent surge in sorghum research is driven by its tolerance to drought / heat stresses and its strong potential as a bioenergy feedstock. Completion of the sorghum genome sequence has opened new avenues for sorghum functional genomics. However, the availability of genetic resources, specifically mutant lines, is limited. Chemical mutagenesis of sorghum germplasm, followed by screening for mutants altered in important agronomic traits, represents a rapid and effective means of addressing this limitation. Induced mutations in novel genes of interest can be efficiently assessed using the technique known as Targeting Induced Local Lesion IN Genomes (TILLING). A sorghum mutant population consisting of 1,600 lines was generated from the inbred line BTx623 by treatment with the chemical agent ethyl methanesulfonate (EMS). Numerous phenotypes with altered morphological and agronomic traits were observed from M2 and M3 lines in the field. A subset of 768 mutant lines was analyzed by TILLING using four target genes. A total of five mutations were identified resulting in a calculated mutation density of 1/526 kb. Two of the mutations identified by TILLING were verified by sequencing in the gene encoding caffeic acid O-methyltransferase (COMT) in two independent mutant lines. The two mutant COMT lines segregate for the expected brown midrib (bmr) phenotype, a trait associated with reduced lignin content and increased digestibility. TILLING as a reverse genetic approach has been successfully applied to sorghum. The diversity of the mutant phenotypes observed in the field, and the density of induced mutations calculated from TILLING, indicate that this mutant population represents a useful genetic resource for members of the sorghum research community. Moreover, TILLING has been demonstrated to be applicable for sorghum functional genomics by evaluating a small subset of the EMS-induced mutant lines.