Research on Arabidopsis thaliana has produced major advances in plant biology because of the many advantages of this system for genetic analysis. But, while it is straightforward to study mutations of Arabidopsis that have obvious, viable, fully penetrant phenotypes, it is still difficult and time-consuming to study mutations that cannot be maintained in homozygous condition, and mutations that have invisible, weak or highly variable phenotypes. This project will produce three resources that facilitate analyses of this large class of mutations: 1) marked chromosome segments, 2) introgression lines and 3) induced polymorphic lines. A marked chromosome segment (MCS) is a chromosome region marked at one end by pNAPIN::dsRED and at the other end by pNAPIN::eGFP. These seed-specific, dosage- sensitive, fluorescent transgenes make it possible to visually identify seeds carrying the marked chromosome segment, as well as seeds homozygous or heterozygous for genes in trans to the MCS. Using an MCS of an appropriate size and location, seeds homozygous or heterozygous for genes/mutations of interest can be accurately identified prior to planting, greatly simplifying genetic and phenotypic studies. MCS lines can also be used to identify recombination events in the vicinity of a gene of interest, facilitating the introgression of mutations and natural polymorphisms into a common genetic background. MCS lines will be generated in both the Columbia (Col) and Landsberg erecta (Ler) ecotypes. Approximately 20 Ler MCS lines covering the entire genome will be introgressed into Col by multiple backcrosses. These MCS introgression lines will be genetically identical to Col, except in the region encompassed by the MCS, and will facilitate the map-based cloning of mutations whose phenotype is only obvious in a Col genetic background. Induced polymorphic (IP) lines offer a similar advantage, but differ from MCS introgression lines in that they involve induced, rather than natural, polymorphisms. IP lines of the Col ecotype will be generated by 3 rounds of sequential mutagenesis, accompanied by selection for lines with a wild-type Col phenotype. IP lines will be made homozygous and the identity of the induced polymorphisms will be determined by next generation sequencing. These new genetic tools will enable a wide variety of experiments that are currently difficult or impossible to perform, adding to the value of Arabidopsis thaliana as a model system for research in plant biology.
Broader Impacts In principle, Arabidopsis thaliana is an ideal organism for teaching plant genetics: it is small, easy to grow, has short life cycle, and its seeds mature in only two weeks. However, its utility as a teaching tool is limited by the difficulty of making controlled intercrosses. Male parents must be emasculated to prevent self-fertilization, and this is difficult to do without practice. This limitation can be overcome by using male sterile mutants, but these mutants must be maintained as heterozygotes, and this introduces significant complications. An MCS stock that makes it possible to unambiguously identify seeds homozygous for the dyt1 male-sterile mutation will be generated, and a laboratory exercise that takes advantage of this stock and transgene insertions created in this project will be developed. Specifically, students will cross plants heterozygous for linked pNAPIN::dsRED and pNAPIN::eGFP transgenes to male sterile plants, harvest the resulting seeds, calculate the recombination distance between these transgene insertions, and generate new MCS lines in the process. A stock containing three linked dominant mutations on the same chromosome as dyt1 will also be produced, enabling teachers to readily generate the plants necessary for a laboratory exercise on three point recombination mapping. These resources will make it possible for students to perform plant genetics in 'real time' in high school or undergraduate courses, and will enable the development of new laboratory exercises that take advantage of the numerous genetic resources available in Arabidopsis.
