Location: Crops Pathology and Genetics Research
2023 Annual Report
Objectives
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.
Progress Report
This is the final report for project 2032-21000-023-000D, Evaluation and Utilization of Novel Genetic Variation in Rice for the Enhancement of Agronomic Performance and Grain Quality, which has been replaced by new project 2032-21000-027-000D, titled “Leveraging Rice Mutant Resources for Trait Discovery, Analysis, and Germplasm Enhancement.” For additional information about this research, please review the new project report.
Induced plant mutants represent a key resource for determining the function of agriculturally important genes and can also serve as genetic resources for developing improved varieties. The overall goal of this project was to identify novel mutations and traits to further our understanding of agronomic performance and grain quality in rice and to develop novel genetic resources for breeding new and improved varieties. There were three objectives: 1) Determine the effect of mutations in key agronomic and grain quality-related genes on traits that affect the production and end-use of rice; 2) Identify the molecular basis of new grain quality and stress tolerance-related traits; and 3) Generate and evaluate populations of mutants with homozygous or “fixed” mutations (i.e., true breeding lines) for agronomic performance and grain quality under field production environments.
In support of Objective 1, ARS researchers in Davis, California, conducted research to characterize the effects of novel mutations in known rice genes affecting some aspect of grain quality. Efforts were focused on starch biosynthesis-related genes and metalloid element (i.e., silicon, arsenic, germanium) transport and accumulation genes. Mutations in the starch branching enzyme I (SBEI) and rice starch regulator 1 (Rsr1) were characterized and their effects on starch biosynthesis and grain traits were investigated using genetics and physico-chemical analyses. Two mutants, NM-4936 (sbeI) and NM-5448 (rsr1), were examined in detail and genetic resources for studies on rice grain quality were developed including double mutants generated by crossing these mutants to another rice grain mutant KDS-1830C. Mutations in three metalloid transport and accumulation genes, low silicon 1 and 2 (lsi1, lsi2) and a rice ATP-binding cassette transporter (OsABCC1) were identified and their effects on the accumulation of silicon and arsenic in rice straw and seeds were characterized. Two lsi1 mutants were found to have significant reductions in silicon content and are being used to investigate silicon-insect interactions with Louisiana State University researchers in Baton Rouge, Louisiana.
In support of Objective 2, ARS researchers identified underlying mutations responsible for novel grain quality, agronomic performance, and stress tolerance-associated traits in rice mutants. Genetic mapping populations were generated for several grain mutants including KDS-1661A, 1830C, 1852, 2173, and NE-334 which were previously identified as opaque, non-waxy endosperm mutants. Mapping populations and double mutants (crosses of two mutans) have been developed and are being used for mutation mapping and characterized for physico-chemical and other grain quality traits (e.g., brewing-related traits). Work with cooperators at Chungnam National University in Daejeon, Republic of Korea, was initiated on mutation discovery using a bulked segregant whole genome re-sequencing approach. This resulted in the identification of the candidate underlying mutation for the KDS-1830C mutant which is in a gene that is likely involved in starch granule organization. Work is underway to confirm these results (see new project). Other work resulted in the identified of altered gelatinization temperature grain mutants, KDS-1623B and 1824B, and the candidate underlying mutation for KDS-1623B was identified in the isoamylase 1 gene. Mapping populations for identifying the KDS-1824B mutation were developed and mutation discovery efforts are ongoing. In addition to grain quality mutants, work towards identifying and characterizing mutations responsible for over a dozen altered cuticle wax mutants was carried out and included the development of several mapping populations. One candidate mutation in the previously reported rice cuticle wax biosynthesis gene OsGL1-1 was identified in the mutant KDS-2249D which is derived from the variety Kitaake. Other mutants in the genetic background of the variety Sabine have been characterized including collaborations to look at the effect of reduced cuticle wax on insect herbivory with Louisiana State University research in Baton Rouge, Louisiana, and fungal disease tolerance with ARS researchers in Stuttgart, Arkansas. Using the same approach as the KDS-1830C mutation detection work, the candidate underlying mutation for the 1558.1/1558.2 (also referred to as SAB-1558) has been identified. Confirmation of this and further characterization of the mutation and mutant is underway (see new project).
In support of Objective 3, research was conducted to screen established rice mutant populations to identify new mutations that impact agronomic performance, grain quality and reproductive cold tolerance. Mutants with alter uptake, transport of nutrients, metabolites, and other compounds affecting agronomic performance and grain quality were identified. The mutant KDS-557B was observed to have increased sensitivity to the phytotoxic metalloid germanium, which had previously been employed to identify metalloid transporter mutants that showed decreased or no sensitivity to germanium. Mapping populations for mutation discovery in KDS-557B were developed and efforts to develop more accurate phenotyping of this germanium hypersensitivity trait are being explored. Research on the evaluation of agronomic and grain quality traits in fixed mutant lines grown under field conditions were limited over the course of this project and focused mainly on generation advance and seed increase of the Kitaake mutant population. Evaluation of the Kitaake mutant lines have been incorporated into the new project. In addition to the Kitaake mutant lines, advanced and early generation mutant lines derived from the varieties Sabine and Nipponbare will be evaluated in the field and seed will be sent to ARS rice gene banks for distribution to interested researchers (see new project).
Accomplishments
1. Mutation underlying novel rice endosperm mutant identified. Rice is unique among major cereals as it is almost exclusively used for direct human consumption as whole milled kernels or table rice, making grain quality traits such as physical appearance of paramount importance. Rice mutants with altered endosperm (i.e., starch) are important for genetic dissection of grain quality and as germplasm for alternative uses (e.g., baking, fermentation). ARS researchers at Davis, California, have identified a mutation underlying a novel rice mutant that forms a central cavity in its endosperm. The mutation is a tool for investigating the processes leading to formation of translucent rice grains while the cavity trait may be useful for fermentation applications. Identification of the mutation provides a perfect marker for selection of the trait and development of germplasm for grain quality research and breeding of rice varieties for brewing.
