Performing Department
Plant Biology
Non Technical Summary
Sexual reproduction in crop plants is typified by the development of true seed as exemplified by seeds borne on infloresences in the grasses, seeds within pods in legumes, and seeds within fruits such as tomatoes, grapes, and cucumbers. In angiosperms, the production of seed involves formation of haploid gametes via meiosis and fusion of egg and sperm nuclei that results in formation of the zygote and subsequently, the embryo. Plants also can reproduce asexually, either through vegetative cuttings, rhizomes, or below-ground storage organs such as tubers, storage roots, corms, and bulbs. These below-ground organs provide a rich source of calories and other nutrients for humans and thus numerous species have been domesticated. Collectively, crops with below-ground organs are referred to as 'root and tuber' crops (e.g. potato, sweetpotato, carrot, and onion). Their importance to agriculture is substantial with 1.9 billion tonnes produced worldwide on 141 million hectares in 2017 (FAOSTAT 2017). These below-ground organs function to provide an asexual reproductive strategy and a mechanism of perennialism for the plant, conferring a greater ability to survive harsh environmental conditions, and a strategy to bypass sexual reproduction. While there are similarities in appearance, tubers, storage roots, corms, and bulbs differ in their origin. Bulbs (e.g., lily, onion) are derived from leaves whereas corms (e.g., gladiolus, crocus) and tubers (potato, yam) are derived from stems; however, differentiation of roots is exemplified by sweetpotato and cassava (storage roots) and carrot (enlarged taproot). In this project, gene regulatory networks will be constructed by combining orthologs identified using comparative genomics (synteny) and phylogenetic analyses with gene co-expression analyses. The resulting networks will then be contrasted to identify gene(s) and modules (sets of interacting genes) that have likely contributed to tuber formation. Key hub genes essential to the trait of tuber development will be functionally validated.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
In this project, gene regulatory networks will be constructed by combining orthologs identified using comparative genomics (synteny) and phylogenetic analyses with gene co-expression analyses. The resulting networks will then be contrasted to identify gene(s) and modules (sets of interacting genes) that have likely contributed to tuber formation. Key hub genes essential to the trait of tuber development will be functionally validated.Objective (1) Using the sister species (potato: tuber-bearing vs tomato: non-tuber-bearing), annotated genome sequence and gene expression profiles from a diverse set of developmental and abiotic/biotic stress conditions will be used to construct gene coexpression networks in each species.Objective (2) Computational approaches including synteny and orthology will be employed to understand the evolution of tuber production at the gene network level and to model core processes in tuber development.Objective (3) From Objective 2, key regulatory genes such as transcription factors will be identified and functionally validated for their role in tuber development using gene editing and over-expression studies in potato and tomato, respectively.
Project Methods
Objective (1). Gene expression profiles from developmental tissues (flowers, fruit, leaves, roots, stems, stolons, tubers, etc) along with a set of leaf and/or root tissue exposed to abiotic (heat, cold, drought) and biotic (methyl jasmonate) from potato (cv. Atlantic) and tomato (cv M82) will be used to construct gene coexpression networks for each species using weighted gene coexpression network analyses (Langfelder and Horvath 2008). As the expression datasets span vegetative, reproductive, and stress conditions, we anticipate capturing the majority of gene coexpression relationships in these two species.Objective (2). A suite of comparative genomics analyses will be performed with the Atlantic and M82 genomes including identification of orthologs and paralogs using Orthofinder (Emms and Kelly 2019) and syntelogs using MCScanX (Wang et al. 2012). Transcription factors will be identified in the Atlantic and M82 genomes using the iTAK software (Zheng et al. 2016). Coexpression modules from Objective 1 will be compared with coexpression modules in tomato to identify key regulator genes in potato (e.g. transcription factors) and cis-regulatory sequences (e.g., promoters and enhancers) that are uniquely associated with tuber formation.Objective (3). To validate the function of candidate genes involved in tuber formation, two approaches will be taken. First, we will use gene editing of the tuber-formation gene in potato using gene editing approaches. In brief, the recently described de novo meristem method (Maher et al. 2020) will be used with a guide RNA designed to the first exon of the targe gene and Cas9 to generate a deletion mutant in Atlantic. Candidate knock-out lines will be tested for their ability to tuberize. Second, we will over-express the candidate gene in M82. Using a constitutive promoter fused to the coding sequence of the putative tuber-formation gene, we will over-express the gene in tomato and score for the presence of tubers similar to that observed for the LOG1 gene (Eviatar-Ribak, et al. 2013). Controls for the functional validation will include gene editing of the maturity locus in potato which is known to control tuberization under long days (Navarro et al. 2011) and over-expression of the LOG1 gene in tomato (Eviatar-Ribak, et al. 2013).Objective (4). A range of species across the angiosperms produce tubers, including yam, potato mint, American groundnut, Jerusalem artichoke, mashua, and oca. Transcriptomes and gene expression profiles for these species will be generated as part of the NSF funded project "Multiple origins of tuber formation: Evolution of a unique storage organ". Using OrthoFinder (Emms and Kelly, 2019), we will identify orthologs of the potato genes that are associated with tuber development in these diverse species. Through correlations with gene expression profiles during tuber development and formation, we will determine whether these orthologs are associated with tuber development in these comparative species that span the angiosperm phylogeny. Third, we will utilize phylogenetic approaches to determine whether these putative tuber-development genes arose from gene duplication and subsequent neo- and sub-functionalization.The Buell lab has extensive experience in comparative genomics, gene expression profiling, and gene coexpression network analysis and thus, do not foresee challenges in completing these objectives. It is possible that candidate tuber-formation genes would be lethal if deleted in potato yet we are able to make an allelic series of knock-out lines to obviate challenges in seeing a phenotype. While over-expression of the LOG1 gene did result in aerial tubers in tomato, it is possible that over-expression of candidate tuber-formation genes fails to yield visible tubers in tomato. The parallel generation of knock-out lines in potato coupled with over-expression in tomato will assist in understanding the function of the candidate tuber-formation genes.