Location: Crops Pathology and Genetics Research
Project Number: 2032-21000-023-000-D
Project Type: In-House Appropriated
Start Date: Feb 13, 2018
End Date: Feb 12, 2023
Objective:
Objective 1: Characterize the effects of novel mutations in known genes on their associated target traits (e.g., seed yield, seed morphology, grain quality, and arsenic accumulation).
Subobjective 1A: Identify phenotypes resulting from mutations in starch biosynthesis- related genes, and determine their effect on grain quality and potential utility for developing new products.
Subobjective 1B: Characterize the effect of mutations in silicon/arsenic transporters on the uptake and accumulation of these elements in rice.
Objective 2: Identify the underlying mutations responsible for novel grain quality, agronomic performance, and stress tolerance-associated phenotypes (e.g., cold, drought, and/or heat) in induced rice mutants.
Subobjective 2A: Identify the mutations responsible for opaque, non-waxy grain phenotypes and evaluate these mutants for alternative uses.
Subobjective 2B: Identify mutations responsible for reduced cuticle wax phenotypes (wax crystal-sparse leaf mutants) and conduct a detailed characterization of the wsl mutants including an evaluation of their performance in the field and response to various biotic stresses.
Objective 3: Screen established rice mutant populations, using forward and reverse genetic approaches, to identify new novel mutations that impact agronomic performance, grain quality, and reproductive cold tolerance in rice.
Subobjective 3A: Identify mutants with altered uptake, transport and accumulation of nutrients, metabolites, and other compounds affecting agronomic performance and grain quality.
Subobjective 3B: Evaluate agronomic and grain quality traits in fixed mutant lines grown under field conditions.
Approach:
Objective 1: Confirm mutations in genes that mediate starch biosynthesis (that were previously identified by the TBS approach) and isolate the homozygous mutant lines if possible. Materials for initial grain quality evaluations will be produced and following sufficient seed production, mutants will be grown and evaluated for the possible effects of the mutations. Mutations will be identified and M4 seeds will be produced prior to the start of this project. M4 individuals (representing all the mutations) will be grown for generation advance and for backcrossing to wild-type Nipponbare to eliminate background mutations. Two rounds of backcrossing will be performed. Additional crosses to generate lsi1/lsi2 double mutants will be performed and mutations of interest (i.e., mutations resulting in reduced grain As) will be used to introgress these mutations into other germplasm.
Objective 2: Using seeds from M3 to M5 generation for each of the mutant lines, two rounds of backcrossing of the mutants to their respective wild-type cultivars will be performed to eliminate background mutations. Progeny of the first backcross will be used for mutation mapping using next-generation sequencing (NGS)-based strategies. Genetic crosses between the mutants to test for allelism and generate double mutants for potential novel phenotypes will be performed as well as crosses to transfer mutant alleles into desired genetic backgrounds (e.g., California varieties). To examine the effects of the reduced cuticle wax on growth, development, and productivity of wsl mutants, field-based evaluations will be conducted. Evaluation of selected mutants for tolerance to two major insect pests of rice, rice water weevil and fall armyworm, will be conducted. Data will be analyzed in the context of the agronomic performance and biotic stress tolerance of the mutants in comparison to wild type to determine if correlations exist and are worth further investigation.
Objective 3: Identify mutants that exhibit either rapid development of Ge-induced lesions or develop lesions but more slowly than wild type. Targeted exon capture and sequencing of the ABC transporter gene family of rice, which consists of 133 members (26) will be performed using a customized MYbaits® sequencing capture kit for NGS, M2 individuals from Sabine, Kitaake and Nipponbare (sibling lines of the M2 individuals forming the rice TBS population) mutant populations will be grown for tissue and seeds. DNA libraries from ~500 M2 individuals (total from the three populations) will be prepared and pooled (pool size 20-30 libraries) for exon capture followed by PCR enrichment of captured sequences and Illumina sequencing. Mutation detection will be performed using the Mutation and Polymorphism Survey (MAPS) pipeline. Additional M2 individuals (~2,000; similar size to the rice TBS population) will be subjected to targeted sequencing depending on the initial results. Candidate mutations will be verified. Candidate mutants for improved cold tolerance at the reproductive stage will be screened.
