DEVELOPMENT OF CHEMICALLY MUTAGENIZED SOYBEAN POPULATIONS FOR FORWARD AND REVERSE GENETICS ANALYSES

Author

EN HUANG, Southern Illinois University CarbondaleFollow

Date of Award

1-1-2009

Degree Name

Master of Science

Department

Plant and Soil Science

First Advisor

Meksem, Khalid

Abstract

Soybean [Glycine max (L.) Merr.] is one of the most economically important crop species in the world. Allelic series that increase genetic variability are very important resources for crop improvement and gene function studies by reverse genetics. Moreover, mutant varieties are not required to comply with the international Genetically Modified Organism (GMO) regulations, which makes them more acceptable to consumers. Two chemical mutagenized soybean populations of approximately 2,260 and 2,450 M2 families from cultivars `Forrest' and `Williams 82' were successfully developed in 2007 ~ 2008. These mutagenized populations with high genetic variability were useful resources for both forward and reverse screening of desired agronomic traits. A large number of visible morphological phenotypes were identified in the mutagenized population, such as albinism, abnormal leaflets and black seed coats, which suggested the success of generating genetic variability by mutagenesis. Additionally, a database was established consisting of the pictures of each plant at maturity, some of the morphological and yield characteristics of each plant. Targeting Induced Local Lesions IN Genomes (TILLING) was used to identify the induced mutations in this project. TILLING combines the advantages of conventional mutation breeding to increase the genetic variability with the high throughput screening of induced mutations for genes of interest. TILLING is a PCR-based method to identify mutations with the aid of the mismatch endonuclease enzyme ENDO1, which detects and cleaves the mismatches between mutant and wild-type DNA amplicons. The presence of a mutation can be visualized using denaturing polyacrylamide gel electrophoresis on a LI-COR 4300 DNA analysis system. The soybean Nodule Autoregulation Receptor Kinase (NARK) gene was targeted in this project to examine the mutation rate of the developed populations. Two mutants in the soybean NARK gene were identified after screening of 768 `Forrest' soybean mutant lines. The mutation rate of the NARK gene in the tested population was 1 mutation/ 540 kb. We obtained this mutation density using EMS at a dose of 0.78% (v/v), which resulted in ~50% seed lethality after treatment. One of the mutants (F262) carried a non-sense mutation in the targeted region of the NARK gene, which resulted in increased nodulation in soybean. Mutant line F262 produced 9 times more nodules than the experiment control. Therefore, TILLING is an efficient approach to identify allelic series in soybean for both gene function studies and crop improvement by providing germplasm with increased genetic variability. Allelic series identified by TILLING are useful resources to link the genotype to particular phenotype.