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Identification and characterization of novel genes contributing to wheat grain yield

Saieed, Md Atik Us (2022) Identification and characterization of novel genes contributing to wheat grain yield. PhD thesis, Murdoch University.

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Grain yield is one of the most important aspects of wheat breeding. Being a polygenic trait, wheat grain yield is regulated by multiple genes and influenced by environmental factors. It is a complex trait which is linked to several traits such as seed number, thousand kernels’ weight etc. The interaction of these yield components with environmental stimulus are poorly understood. In the current study, to improve our understanding, phenotypic plasticity of contributing traits to the grain yield was explored. The phenotypic plasticity is the variations in the expressed phenotype by an individual genotype under environmental influences. The experiment consisted of 225 Westonia-Kauz double haploid (DH) lines and evaluated in five environmental conditions. The result demonstrated that, across the DH lines, the spikelets/spike was the most plastic trait. The least plastic character was the grain protein content. Yield plasticity was found higher at favourable conditions. An increase in yield plasticity by 0.1 units was associated with an increase in maximum yield by 4.45 kg ha−1 (p≤0.001). The generated knowledge regarding trait plasticity will be useful in dissecting the genetics for yield improvement particularly at the situation of rapid climate change. Identifying quantitative trait loci (QTL) and incorporating them in the breeding program has been a widely used approach for genetic improvement of yield and its components. QTL mapping suggests a considerable size of chromosomal location harbouring genes contributing to the trait which also contains many non-target genes. Thus, a more precise identification of contributing gene would be much helpful for an efficient breeding approach. However, functional confirmation of each individual gene of a QTL region is quite laborious and expensive work. In-silico approach provides the opportunity to reduce the down-stream workload by reducing the number of candidate genes in a systematic approach. Apart from the trait plasticity research, the current study also used a pipeline combining bioinformatics and laboratory approaches to identify the contributing genes of a grain yield QTL from a double haploid (DH) population of Westonia × Kauz. Assembling the QTL region on the International Wheat Genome Sequence Consortium (IWGSC) whole-genome sequence using the flanking 90K SNP markers identified the genomic region of 20 Mbp. Gene annotation revealed 16 high confidence genes and 41 low confidence genes in that genomic region. Further functional gene annotation, ontology investigation, pathway exploration, and gene network study using publicly available expressional data enabled short-listing of four genes for down-stream functional confirmation. Complete sequencing of those four genes demonstrated that only two genes namely ferredoxin-like protein and tetratricopetide-repeat (TPR) protein gene are polymorphic between the parental cultivars. Two single nucleotide polymorphism (SNP) variations were observed in the exon for both genes, and one SNP resulted in changes in amino acid sequence. The qPCR-based gene expression showed that both genes were highly expressed in the high-yielding double haploid lines. In contrast, gene expression was significantly lower in low-yielding lines. Results indicate that these two genes are potentially the underlying genes for the grain yield QTL. To investigate the association of the selected genes with grain yield and yield components at a wider level, further genetic and phenotyping experiments were conducted on a set of 143 historical wheat cultivars of Australia. For both genes, the identified alleles in the parental cultivars have been named as Westonia and Kauz allele. Characterising the allelic composition of the genes demonstrated that, for ferredoxin gene, 34.9% cultivars possessed Westonia allele and 16.9% cultivars possessed Kauz allele. In case of TPR gene, 20.9% cultivars possessed Westonia allele and 23.8% cultivars possessed Kauz allele. For both genes, cultivars having Westonia allele showed significantly higher seed width, thousand kernels’ weight and grain yield at different environmental conditions which clearly indicated that these genes are playing important roles in determining grain yield. For further level of functional confirmation, CRISPR-Cas9 based genome editing experiment was carried out on the TPR gene in Arabidopsis using orthologous gene. Agrobacterium mediated floral dip transformation was performed using immature inflorescence containing Cas9 gene. Knock-out mutants were selected by sequencing the target gene. Phenotypic data were collected from T2 generation on leaf length, stem length, number of branches on the main stem, days to flowering, days to maturity, pods/plant, and pod length. A significant reduction was observed in pods/plant, leaf length, and days to flowering and maturity. Gene expression analyses was performed on the selected genes responsible for increased seed size, seed number, and vegetative growth, in transgenic lines of Arabidopsis. Significant reduction in gene expression was observed for ARGOS, GRF1 and GW2 genes suggesting the role of TPR gene in downregulating essential growth regulator genes and its involvement in grain yield indirectly. Overall, this study demonstrated successful use of multiple research approaches in identification of a novel candidate genes of a yield related QTL. This approach can be utilised in exploring the candidate genes of other QTLs. The identified novel genes demonstrated the potential of improving the wheat grain yield which might be included in the breeding program for further yield improvement.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): Food Futures Institute
State Agricultural Biotechnology Centre
United Nations SDGs: Goal 12: Responsible Consumption and Production
Supervisor(s): Ma, Wujun and Islam, Shahidul
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