9:00 am - 10:00 pm
Event details: A graduate exam seminar is a presentation of the student’s final research project for their degree.
This is an ALES MSc Final Exam Seminar by Mohammed Mukthar. This seminar is open to the general public to attend.
MSc with Drs. Stacy Singer and Hari Poudel.
Zoom Link: https://ualberta-ca.zoom.us/j/98824752732?pwd=EFDazT9pciI3CUi0jyxFMDjAkLAdSd.1
Thesis Topic: Enhancement of Total Shoot Lipid Content of Alfalfa and Sainfoin Using Chemical Mutagenesis and CRISPR-Cas9-Mediated Genome Editing
Abstract:
Methane (CH4) emissions from cattle, resulting from the inefficient fermentation process in the ruminants, is one of the major contributors to anthropogenic greenhouse gas emissions. One strategy to reduce CH4 emissions from ruminants is to increase the energy density of their feed through lipid supplementation. However, this approach is often costly and sometimes impractical. In the present day, available cultivars of forage crops such as alfalfa (Medicago sativa), a notable legume forage in terms of ruminant rations and export value, and sainfoin (Onobrychis viciifolia Scop.), a bloat-free forage, contain only ~1-3% lipid on a dry matter (DM) basis. As such, the overall aim of this study is to improve the total shoot lipid content (TSLC) of these crops using random and targeted mutation.
The first objective of this study is to generate the first generation of chemically mutagenized alfalfa and sainfoin populations with increased TSLC. M1 populations, including approximately 500 plants from alfalfa (cv. AC Blue J) and sainfoin (cv. AAC Mountainview), respectively, were established in the greenhouse following treatment with 0.5-1% ethylmethane sulfonate (EMS). TSLC of samples from these M1 populations were analyzed using near-infrared spectroscopy (NIRS) and validated through Gas Chromatography-Mass Spectrometry (GC-MS). After a series of selections, approximately 16 -18 mutants with high (a relative increase of 12.3 – 12.8 % lipid on a DM basis compared to control) or low (a relative decrease of 10.6 – 11.5% on a DM basis compared to control) lipid contents were selected from M2 populations for morphological studies and advancement of M3 generations (Chapter 2).
Subsequently, RNA sequencing was performed on two selected genotypes from each category (low-lipid, control, high-lipid) to identify genes associated with lipid biosynthesis in leaf and stem tissues (Chapter 3). In leaf tissue, the analysis revealed 512 differentially expressed genes (DEGs) in the high-lipid vs. control comparison, 210 DEGs in the low-lipid vs. control, and 362 DEGs in the high vs. low lipid comparison. Stem tissue exhibited a greater number of DEGs, with 657 DEGs in high-lipid vs. control, 166 DEGs in low-lipid vs. control, and 518 DEGs in high vs. low comparisons. Bases on the MapMan analysis, four lipid-related genes were identified in the high-lipid mutants, including biotin carboxyl carrier protein (BCCP), beta-ketoacyl-acyl carrier protein synthase III (KAS III), CER2 protein, and lipase-like protein, based on differential expression comparisons of high-lipid mutants vs. both control and low-lipid mutants. In contrast, three lipid-related genes were identified in the low-lipid mutants (compared to control), including At2g26170 (cytochrome P450, family 711, subfamily A, polypeptide 1), lipase-like protein, and BCCP. The lipid-related DEGs identified in this study provide valuable targets for genomic breeding aimed at enhancing lipid content in the vegetative tissues of forages.
The second objective of the study is to enhance TSLC in alfalfa by downregulating PEROXISOMAL ABC TRANSPORTER 1 (PXA1) and SUGAR DEPENDENT 1 (SDP1) genes, which are involved in lipid breakdown, using the Clustered Regularly Interspaced Palindromic Repeat (CRISPR) and CRISPR Associated Protein 9 (Cas9) genome editing tool (Chapter 4). Gene editing frequency droplet digital PCR (GEF-ddPCR) assays and Sanger sequencing were employed to confirm the presence of mutations at the target sites. Eight unique PXA1-edited and 11 unique SDP1-edited genotypes were identified, with gene editing frequencies ranging from 25% to 75%. Lipid content analysis revealed that both PXA1 (four mutants with increased TSLC, one mutant with increased leaf lipid content, and four mutants with increased stem lipid content) and SDP1 (four mutants with increased leaf lipid content, and one mutant with increased stem lipid content content) edited genotypes exhibited significant disruptions in the lipid accumulation and fatty acid composition. There were no consistent morphological variations in either the PXA1 and SDP1 mutants, with the exception of poor morphological traits observed in SDP1-gRNA3 mutants and consistent delayed flowering in all SDP1 mutants.
In conclusion, this study explores the use of EMS-mediated mutagenesis and CRISPR-Cas9 editing to improve TSLC in forage legumes like alfalfa and sainfoin. The research highlights the potential to reduce CH₄ emissions, improve animal performance, and enhance the quality of ruminant feed. Key genes involved in lipid metabolism were identified, and further work is needed to optimize genetic modifications. The resulting germplasms will provide high-quality feed and benefit both producers and consumers.
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