Various approaches can be implemented to identify a gene responsible for a phenotype of interest due to mutagenesis. Current next generation sequencing (NGS) technology allows whole genome sequencing of a mutant and accelerates the identification of mutation-induced polymorphisms in mutagenised organisms. In the model legume Lotus japonicus ecotype Miyakojima (MG-20), an abscisic acid (ABA) insensitive mutant called Beyma was previously isolated by ethyl methanesulphonate (EMS) mutagenesis and originally identified as a heterozygous dominant mutation. Beyma is slow-growing, wilty and incapable of regulating stomatal opening. A wild type segregant of the Beyma mutant (WTS) was also isolated from a self-generation of heterozygous Beyma mutants. ABA plays numerous roles in plant growth and development as well as morphogenetic responses including nodulation in legumes. Yet, there is a lack of ABA studies in legumes. Therefore, the Beyma mutant in L. japonicus allows a wide range of studies that will provide in-depth information of ABA signaling in nodulation as well as stress responses in legumes.
This project presents an attempt to identify a causal gene in the ABA insensitive Beyma using the NGS technology. Tissue from a homozygous Beyma mutant, WTS and MG-20 wild type (WT) plants was subjected to the whole genome sequencing, generating about 300 million paired end of short-sequence reads. The Kazusa MG-20 genome was used as reference for read mapping. Single nucleotide polymorphisms (SNPs) were called based on mutations in the Beyma and WTS genomes as compared to the re-sequenced MG-20 genome. As a preliminary study, three procedures of read mapping and variant calling were performed to undertake a genomic comparative analysis and identify the causal gene.
Sequencing of single genomes of the three plants showed a mutation occurred in every 208 kb (WTS) and 202 kb (Beyma) with a bias mutation of G/C-to-A/T changes at low percentage. Most mutations were intergenic. The mutation spectrum of the genomes was comparable in their individual chromosomes but each mutated genome has unique alterations, which are useful to identify causal mutations for their phenotypic changes. A total of 59 SNPs were identified as potential putative causal Beyma mutations, which were located in various annotated genes in the MG-20 genome. Verification of these mutations could not be done due to time constraint but will be performed in future. A candidate gene ii approach was also carried out by selecting ABA-related genes based on their roles in ABA biosynthesis to signalling, directly or indirectly. Mutations were found in loci of ABA INSENSITIVE (ABI) 1, ABI2, HAB1, HAB2, ABI3, ABI4, and ABSCISIC ACID 8'-HYDROXYLASE 2 in both mutant genomes or only in the WTS genome. Unique mutations also occurred in EARLY RESPONSIVE TO DEHYDRATION 7 and ABSCISIC ACID 8'-HYDROXYLASE 1/ P450 CYP707A1 genes, which were predicted to be impaired in their downstream regions. Although the candidates were not affected in the essential region of the genes, the candidate gene approach has eliminated all the candidates as the putatively causal Beyma gene.
In order to intensify the identification of the causal Beyma gene, re-sequencing of the Beyma and WTS genomes was performed on pooled DNA. In this analysis, the presence of mutations was more frequent in both mutagenised genomes (~18-35% increase), resulting in higher rate of base changes and demonstrated that pooled DNA sequencing increased the mutation frequency. In addition, 69 unique Beyma SNPs were predicted to cause nonsynonymous changes and will be verified in future study. Nevertheless, a mutation (locus named chr3.CM0451.1060.r2.d) was found in both batches of sequencing. It was a C-to-T mutation, which changed glutamic acid to lysine in an F-box family gene. This gene could be the Beyma gene but it requires verification.
In conjunction with the genome sequence analysis, other analyses were also done to prepare plant materials for sequencing and future verification. Plants were subjected to ABA treatment on seed germination and root development to select good mutant lines and WTS plants. Outcross between Beyma and L. japoncus ecotype Gifu was also performed for the segregation analysis of the putative causal SNPs in the F2 plants carrying homozygous WT alleles. This project highlighted the overall molecular changes produced in the whole genome of MG-20 mutants due to EMS mutagenesis. In future, the identification of the causal Beyma gene will possibly show a novel gene involved in ABA sensitivity in legume systems. In addition, it should be of great interest for researchers in forward genetics in legume studies.