Document Type

Thesis - Open Access

Award Date

2020

Degree Name

Master of Science (MS)

Department

Biology and Microbiology

First Advisor

Donald Auger

Keywords

Ae1-5180, Deletions, Mu1, Mutation, r1 gene, starch branching enzyme IIb (SBEIIb)

Abstract

This thesis consists of two independent projects dealing with two different aspects of maize. The first project is about the maize reproduction, where we try to uncover the candidate genes of two mutations that affect pollen viability and successful fertilization. The second project is about maize endosperm starch, where we developed molecular markers to characterize a mutant allele that knocks a major starch branching enzyme in maize.

Analysis of two maize mutations that arose after exposure to radiation from a plutonium-beryllium source
Maize is a monoecious and diclinous plant. Sexual reproduction takes place by the fusion of haploid male and female gametes produced in the anther and ovule, respectively. The male gametophyte is pollen, a trinucleate structure, that consists of two sperm cells and a vegetative cell. The female gametophyte is embryo sac, that consists of an egg cell, two synergids, two central cells and a variable number of antipodal cells. After pollination, the vegetative cell germinates and forms a pollen tube. The pollen tube elongates through the style, penetrates the ovule, and finally burst after reaching embryo sac to release two sperm cells. One sperm cell fuse with the haploid egg cell in embryo sac and forms a diploid embryo, whereas the other fuses with two central cells to form a triploid endosperm. In the first project, we developed two mutant lines, PB1 and PB2 by exposing kernels heterozygous for r1 (colored 1) and c1 (colored-aleurone 1) color factors to a fast neutron source for three and six months, respectively. Two mutagenized plants when crossed with r1 and c1 testers, showed reduced transmission for the R1 marker through pollen, but normal transmission through female. Instead of 50%, the R1 transmission rate through pollen was 23.6% ±0.61% (mean ±se) for PB1, and 1.48% ±0.44% (mean ±se) for PB2. Comparison of whole genome sequences of PB1, PB2 and the unmutated parental line PB1, shows that 32 genes are affected by the deletions in PB1, whereas in PB2, 45 genes are affected. In PB2, genes that code for mechanosensitive ion channel protein, C2H2 type Zinc finger family protein and β-expansin have been previously reported to affect pollen tube germination and elongation, which makes them good candidates.

Developing molecular markers for antimorph Ae1-5180, an alternative to ae1 to increase amylose content in maize endosperm
Maize endosperm consists of mostly starch, which is made of unbranched amylose and branched amylopectin residues. In most Midwestern dents, the endosperm starch is about 75% amylopectin and 25% amylose. Branch formation is catalyzed by starch branching enzymes. Starch branching enzyme IIB (SBEIIb) encoded by ae1 (amylose extender 1), is the primary starch branching enzyme that forms amylopectin. The amylose content increases up to 50% when ae1 is homozygous recessive. An antimorph allele (Ae1-5180), was identified that acts in a dominant fashion to eliminate SBEIIb (Stinard et al., 1993). The essential portion of this mutant allele is a 3kb XhoI fragment that includes Mutator1 (Mu1) element flanked by an inverted duplication of the ae1 gene. Based on the restriction map (Stinard et al., 1993) we developed an approximate predicted sequence for Ae1-5180 using the known sequences of the wildtype ae1 gene and Mu1 transposon. Using this, we designed PCR primers targeted to amplify regions within a 3 kb XhoI restriction fragment that appears crucial for the dominant action of Ae1-5180. The primers amplify two DNA bands from samples expected to have Ae1-5180, but none from B73. The sequence of the lower molecular weight band matches with 181 bp of Mu1 and 364 bp of Ae1 (545 bp). The higher molecular weight band also possesses 181 bp of Mu1 but includes 438 bp of Ae1 (619 bp). The additional 74 bp includes a restriction site for NotI, which was predicted from the original restriction map. Sequencing results indicate that these primers reliably detect the presence or absence of the Ae1-5180 allele. Also, the insertion site of Mu1 in Ae1-5180 is now exactly defined.

Format

application/pdf

Number of Pages

92

Publisher

South Dakota State University

Rights

In Copyright - Educational Use Permitted
http://rightsstatements.org/vocab/InC-EDU/1.0/

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