Document Type

Thesis - Open Access

Award Date

2020

Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology

First Advisor

Wanlong Li

Keywords

CRISPR-Cas9, miR156, plant architecture SPL, wheat, yield

Abstract

Higher and stable yield is always a major objective of wheat genetic improvement programs. The SQUAMOSA-promoter binding protein-like (SPL) genes constitute a small family of plant-specific transcription factors with diverse functions in plant development and growth and have great potential in improving yield and other major agronomic traits. However, the functional characterization of the SPL gene family in wheat is far behind other cereal crops such as rice. Using phylogenetic analysis, we identified 56 wheat orthologues of rice SPL genes belonging to 19 homoeologous groups. Among these 19 orthologous TaSPL genes, nine harbor the micro RNA 156 recognition element (MRE) in their last exon except for one gene, TaSPL13, which harbors the MRE in 3’-untranslated region (UTR). We edited the MRE of TaSPL13 using CRISPR-Ca9 and generated 11 mutations in three homoeologous genes. CRISPR mutations of 10- and 25-bp deletions in MRE increased the expression of TaSPL13-A by ~1.7 and 1.9-fold, respectively, and two 1-bp insertion mutations in MRE upregulated the expression of TaSPL13-B by 2.25-fold compared to wildtype (WT). Phenotypic evaluation showed that the 1-bp insertion mutations in TaSPL13-B has the highest impact on decreasing days to flowering, tiller number, and plant height, and has positive effects on grain size and grain number. In terms of flowering time, the expression of wheat Vernalization 1 gene, which is orthologous to flowering gene APETALA 1 of Arabidopsis, was upregulated by 2.3-fold in the double mutant TaSPL13-ab, which combined the 5-bp deletion in TaSPL13-A and the 1-bp insertion in TaSPL13-B. Our results demonstrate the pleiotropic effects of TaSPL13 mutants in wheat and functional conservation among the orthologous SPL gene between wheat and the model plants Arabidopsis and rice. The TaSPL13 holds great potential in improving wheat yield by simultaneously increasing grain size and number. The novel mutations generated in the homeologs can be utilized in wheat breeding programs to improve these agronomic traits. While working with the CRISPR-Cas9 technique we developed a new and simple protocol for cost-effective genotyping of CRISPR mutations. Simultaneously, we discuss the advantages and pitfalls of various genotyping platforms such as mismatch cleavage assay, restriction enzyme assay, ribonucleoprotein assay, and Sanger sequencing. The case study-based approach provides details about the methods and can act as a guide to screen CRISPR mutations in wheat.

Library of Congress Subject Headings

Wheat -- Genetics.
CRISPR (Genetics)
Wheat -- Yields.
Protein binding.

Format

application/pdf

Number of Pages

172

Publisher

South Dakota State University

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Rights Statement

In Copyright