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Research Project: Evaluation and Utilization of Novel Genetic Variation in Rice for the Enhancement of Agronomic Performance and Grain Quality

Location: Crops Pathology and Genetics Research

2021 Annual Report

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.

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
In support of Objective 1, research continued on characterizing the effects of novel mutations in known genes on seed yield, seed morphology, grain quality, and accumulation of metalloid elements (e.g., silicon, arsenic) in rice. Due to a critical vacancy and maximized telework, efforts were limited to evaluation of two F2 mapping populations derived from the mutant NM-5448, which harbors a mutation in the rice starch regulator 1 [rsr1] gene and exhibits larger grains but none of the previously reported effects on the grain starch quality. As reported in fiscal year (FY)20, this mutant was crossed to the varieties Nipponbare (NNM-5448’s wild-type progenitor) and Kitaake. Seeds of F2 plants from each population are being evaluated for seed size and have been planted in FY21 to produce the next generation (F3). This generation advance process will continue for several more generations to produce lines that stably express traits resulting from the mutation. The goal of this research is to determine if the rsr1 mutation is responsible for the larger grain size in NM-5448 and to generate materials for use in evaluating starch gene expression and quality-related traits and for breeding. Due to continued maximized telework and a critical vacancy, double mutant analysis involving the crossing NM-5448 and the previously characterized NM-4936 mutant (starch branching enzyme 1 gene mutant) and NM-5448 and the mutant KDS-1830C (see Sub-objective 2A) has been delayed until FY22. In FY21, elemental analysis of straw samples from 13 mutants harboring mutations in transport and accumulation genes Lsi1 (three), Lsi2 (nine), and OsABCC1 (one) was conducted. Analysis of the data is currently underway to determine if the uptake and accumulation of other metalloid elements besides silicon and arsenic are affected in these mutants. Selected mutants are being grown at a new field site to produce seeds for more analysis and for use in studies to examine the effect of reduced silicon content on the ability of some of these mutants to tolerate pests and pathogens of rice. A subset of the low silicon mutants has also been provided to cooperators to characterize their resistance to insect pests. All the mutants are also being grown in the greenhouse for backcrossing to the wild-type progenitor variety Nipponbare and to other varieties to transfer these mutations into different genetic backgrounds for study and possible use in breeding. In support of Objective 2, research continued on advancing the analysis of the KDS-1830C grain mutant. Populations derived from crossing the KDS-1830C mutant with wild-type varieties Kitaake (its progenitor), California rice variety M-103, and rsr1 mutant NM-5448 are in various stages of characterization. In FY21, F3 seeds from the F2 populations that were generated by crossing with Kitaake and M-103 were planted for generation advance and to confirm the phenotyping of the internal cavity trait of KDS-1830C. Generation advance of lines derived from a cross between the endosperm/starch mutants KDS-2173A and KDS-1852 was continued using F4 seed of the sibling lines K7352-49.1.1, K7352-49.1.3, and K7352-49.3.2. The original line K7352-49 was selected as it exhibited a high proportion of grains with a white core endosperm trait that is a characteristic of rice varieties used for brewing of premium quality sake (i.e., Japanese rice wine). This line will be advanced to the F8 or greater generation with selections at each generation for the white core trait while maintaining the other traits (e.g., tiller number, height, flowering time, fertility) of the Japanese variety Kitaake from which the parents KDS-2713A and 1852A are derived. In FY21, seed stocks of one of more of these lines will be generated for use in field planting in FY22 for the production of sufficient quantities of rice for preliminary sake brewing evaluations. Work on the grain mutants KDS-1661A.3 and NE-334.1 and identification of the mutations underlying all the grain mutants has been delayed. In FY21, work continued on the development of a Kitaake line with the KDS-2249D mutant allele of the OsGL1-1. Seeds produced from the self-fertilization of a single plant derived from a cross of the line DK3-128.4 (itself derived from a cross of KDS-2249D and Kitaake) by Kitaake were planted and individuals exhibiting the wax crystal-sparse leaf (2249D mutant phenotype) were identified and allowed to self-fertilize to produce seeds and crossed to Kitaake again to further reduce the presence of backgrounds mutations. This cross produced two plants that appeared to be wild-type and therefore representative of true crosses. The self-seed of these plants were harvested and about 50 seeds were planted and phenotyped. Nine individuals were identified in this population as exhibiting the mutant phenotype and seeds have been collected from these plants for use in studies to examine responses to abiotic and biotic stresses and for preliminary phyllosphere microbiome surveys, which are now planned for early FY22. Work continued on the genetic analysis and characterization of the cuticle wax-deficient Sabine mutants 6-1A-3, 7-17A-5, 11-39A-1 and 1558.1. The research on the response of three of these mutants (6-1A-3, 7-17A-5, and 11-39A-1) to the insect pests, fall armyworm and rice water weevil, was published in FY21. Wax content analysis of the 1558.1 mutant was completed in FY21. This mutant has larger, but fewer epicuticular wax crystals compared to wild-type Sabine and the gas chromatography/mass spectrometry analysis indicates that the 1558.1 mutant has elevated levels of alkanes and ketones. Backcross lines (BC1F2) exhibiting the mutant phenotypes of the Sabine 6-1A-3, 7-17A-5, 11-39A-1, and 1558.1 mutants were sent to ARS colleagues for evaluation of resistance to fungal pathogens. In addition, mutant lines 11-39A and 1558.1 were provided to cooperators in California to re-test their response to stem rot fungus this year. Results in FY19 suggested that 1558.1 was highly tolerant to stem rot disease and no testing was performed in FY20. Work to identify the mutations responsible for the cuticle wax-deficient trait is now underway for 1558.1 and the three other mutants will be examined in early FY22. In FY21, twelve other cuticle wax-deficient mutants are being grown for backcrossing to Sabine. This will facilitate the production of genetic mapping populations to identify the mutations underlying these mutants and lines that will be used to evaluate the effect of each mutation on the response of the plants to the environment (i.e., abiotic and biotic stresses). In support of Objective 3, research continued on screening of rice mutant populations to identify novel mutations that impact agronomic performance, grain quality and reproductive cold tolerance in rice. Due to maximized telework and a critical vacancy, the work in FY21 involved processing seeds from the Sabine mutant population (M4 generation) and planting of mutant lines (various generations) from the Kitaake, Nipponbare, and Sabine populations for exon capture and sequencing and for generation advance and seed amplification. Pandemic-related issues and critical staffing issues of our university hosts affected greenhouse services resulting in delayed planting. Approximately 500 M7 generation Kitaake mutants are being grown this year to produce M8 seeds for field-based seed increase and evaluation. Work on the Sabine (M4 generation) and Nipponbare (M3 generation) populations has been de-prioritized due to limited resources. In addition, M2 seeds from Sabine, Kitaake, and Nipponbare mutant populations are being grown for exon capture and sequencing experiments and for M3 seed production. Efforts are underway to identify a new site for field-based research due to insurmountable obstacles in using our previous field location on the university campus as detailed in FY20. In addition to advancing the mutant populations, work on the genetic and physiological characterization of the germanium tolerance mutants continued. F3 seeds from an F2 mapping population derived from a cross between the germanium hypersensitive Kitaake mutant KDS-557B and wild-type Kitaake have been planted for generation advance and are also being used to confirm phenotyping of the F2 individuals in FY19. Generation advancement to the F5 or later generation will allow more accurate phenotyping of the germanium hypersensitivity trait and assist in the identification of the mutation underlying KDS-557B. Work on other Kitaake mutants showing altered responses to germanium and the screening of a Sabine mutant population for additional germanium tolerance mutants has been de-prioritized.

1. Rice mutants uncover role of epicuticular waxes in resistance to insect pests. Epicuticular waxes are the outermost protective barrier of land plants but their involvement in rice-insect pest interactions is unknown. ARS researchers in Davis, California, in cooperation with Louisiana State University researchers in Baton Rouge, Louisiana, showed that epicuticular wax-deficient mutants of rice are less resistant to two major insect pests, rice water weevil and fall armyworm. These mutants are valuable genetic resources for studying the role of epicuticular waxes in biotic stress tolerance in rice. This knowledge may lead to new strategies for developing insect resistant rice varieties.

Review Publications
Bernaola, L., Butterfield, T.S., Tai, T., Stout, M.J. 2021. Epicuticular wax rice mutants show reduced resistance to rice water weevil (Coleoptera: Curculionidae) and fall armyworm (Lepidoptera: Noctuidae). Environmental Entomology. 50(4):948-957.
Jeon, Y., Lee, H., Kim, S., Shim, K., Kang, J., Kim, H., Tai, T., Ahn, S. 2021. Natural variation in rice ascorbate peroxidase gene APX9 is associated with a yield-enhancing QTL cluster. Journal of Experimental Botany. 72(12):4254-4268.