Doctoral Dissertations

Rekha Prabhu

Date of Award

8-1995

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Life Sciences

Major Professor

Peter M. Gresshoff

Abstract

This study dealt with the molecular genetic analysis of soybean (Glycine max (L.) Merr.) using predominantly single primer amplification markers. The long term aim was to provide additional information needed for the cloning of genes involved in the process of nodulation in legumes. Initial emphasis was given to the mutation nts382 which confers supernodulation and the lack of autoregulation to soybean. The nts mutation as well as the onset of autoregulation are not associated with major polypeptide changes in the leaves of soybean. DNA amplification fingerprinting (DAF) using high primer to template ratio and single, very short arbitrary primers, was used to generate DAF markers in soybean (Glycine max (L.) Merr.). The amplification reaction was carried out with soybean genomic DNA and 8 base long oligonucleotide primers. The DAF reaction was optimized for template and enzyme concentration. Silver stained 5% (w/v) polyacrylamide gels containing 7 M urea detected between 11 and 28 DAF products with primers of varying GC content (ranging from 50-100% GC). Depending on the intensity, DAF markers were classified into three classes. The intensity of silver-stained DNA fragments was linear from 150 pg to 5 ng/band and did not show any abnormal mobility for size or amount of DNA in each band. DAF profiles were reproducible for different DNA extractions and gels. Forty markers were detected by 26 primers when comparing Glycine soja and Glycine max. The inheritance of DAF markers was studied using a cross between the ancestral Glycine soja PI468.397 and Glycine max (L.) Merr. line nts382, F1, and F2 progeny. Most DAF markers were inherited as dominant Mendelian markers in F1 and F2 populations. However, abnormal inheritance occurred with about 25% of the polymorphisms. One marker was inherited as a maternal marker, presumably originating from organelle DNA while another showed apparent paternal inheritance. To confirm the nuclear origin and utility of dominant Mendelian markers, three DAF polymorphisms were mapped using a F11 mapping population of recombinant inbred lines from soybean cultivars 'Minsoy' x 'Noir1'. The utility of DAF markers was also demonstrated for pedigree assessment and cultivar identification. DAF data produced a dendrogram similar to that produced by RFLP and pedigree data. Cloning and hybridization of DAF products showed that DAF polymorphic bands may contain sequences of multiple origin. The study showed that DAF- generated polymorphic markers occur frequently and reliably, that they are inherited as Mendelian dominant loci and that they can be used in genome mapping.

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