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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

2020 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
Objective 1 involves research characterizing the effects of novel mutations in known genes on their associated target traits (e.g., seed yield, seed morphology, grain quality, and arsenic accumulation). Research in support of Sub-objective 1A progressed in determining whether mutations in starch biosynthesis-related genes have any effect on grain quality. Due to a critical vacancy and maximized telework, efforts were limited to seed processing and phenotyping of two F2 mapping populations derived from the mutant NM-5448 (mutation in the rice starch regulator 1 [rsr1] gene and exhibiting larger grains) and Nipponbare (wild-type progenitor) and Kitaake. Kitaake is a variety with similar grain attributes to Nipponbare, but which is very early maturing. Completion of the seed phenotyping and work to develop recombinant inbred line mapping populations is delayed until early FY21. 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 maximized telework, 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) is now delayed until FY21. In support of Sub-objective 2B, analysis of total arsenic in straw and grain samples from 13 mutants harboring mutations in transport and accumulation genes Lsi1 (three), Lsi2 (nine), and OsABCC1 (one) was completed. Mutants were grown in two sites (Davis and Biggs, California) in 2018. Analysis of total arsenic content in straw and grain revealed some differences between the two locations. Mutant NM-4903 showed reduced total grain arsenic in the Davis but not the Biggs location. Backcrossing of mutants to the wild type progenitor Nipponbare to remove background mutations may be needed to clarify the impact of the specific mutations in the Lsi1, Lsi2, and OsABCC1 genes on silicon and arsenic content. Some of this work was delayed in FY19 and due to a critical vacancy and maximized telework will be delayed until FY21. Analysis of progeny from crosses made between mutants in FY18 has similarly been delayed. Objective 2 focuses on identifying 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. In support of this Sub-objective 2A, grain trait data continues to be collected from F2 mapping populations from genetic crosses of opaque grain mutants. The primary focus in FY20 has been on advancing the analysis of the KDS-1830C mutant. Populations derived from crossing the KDS-1830C mutant with wild-type varieties Kitaake (its progenitor) and M-103 as well as other mutants (NM-5448 and KDS-1824B, an altered gelatinization temperature mutant) are in various stages of characterization. In FY19, it was observed that some of the F2 progeny from a cross between KDS-2173A and KDS-1852 produced F3 seeds with a white core trait associated with premium sake brewing rice varieties. The F3 seeds of selected F2 plants are being advanced to produce true breeding lines (F7 or F8 generation) for seed production in the quantities needed for evaluating suitability for brewing. The early maturity/short duration of these lines, which are derived from the Kitaake variety, should help in reducing the time to produce fixed/true breeding materials (2-3 generations per year). In FY20, characterization of two altered gelatinization temperature mutants KDS-1623B and KDS-1824B was described including the identification of a mutation in the isoamylase 1 (starch debranching enzyme) gene as the basis for the KDS-1623B mutant phenotype. Work identifying the cause of the endosperm mutants (Sub-objective 2A) was delayed until FY21 due to maximized telework and a critical vacancy. In support of Sub-objective 2B, 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. Seeds from plants exhibiting the 2249D mutant phenotype and derived from one (FY19) and two (FY20) backcrosses with wild-type Kitaake were collected for use in phenotypic evaluations including responses to abiotic and biotic stresses and for preliminary phyllosphere microbiome surveys. These experiments have been delayed until FY21 due to maximized telework in FY20. Work continued on the genetic analysis and characterization of the Sabine wsl mutant 6-1A-3, 7-17A-5, 11-39A and 1558.1. In FY20, wax compositional analysis and quantification of the 6-1A-3, 7-17A-5 and 11-39A mutants was completed and results are included in a manuscript being submitted with cooperators in Louisiana who have evaluated the susceptibility of these three wsl mutants to the insect pests fall armyworm and rice water weevil as reported in FY19. Initial wax analysis of the 1558.1 mutant was conducted in FY20 but completion of this characterization has been delayed until FY21. In FY19, F2 populations derived from backcrossing the four Sabine wsl mutants with wild-type Sabine were phenotyped resulting in confirmation of a single gene mutation mode of inheritance for each mutant. A small number of F2 individuals exhibiting the mutant (wsl) phenotype from each population was selected to produce F3 seeds. These lines, which have fewer background mutations than the original mutants, will be used for characterization studies and additional genetic crosses in FY21 due to maximized telework in FY20. The F2 populations derived from the four wsl mutants were intended for mutation mapping; however, maximized telework and a critical vacancy in FY20 required postponement of this work until FY21. Preliminary field-based evaluation of tolerance of mutant lines 11-39A and 1558.1 to stem rot fungus (S. oryzae/M. salvinii) with cooperators in California suggested that 1558.1 was highly tolerant to stem rot disease. Further studies to examine this result are planned for FY21. Objective 3 focuses on screening 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. In support of Sub-objective 3A, research continued on the genetic and physiological characterization of germanium tolerance mutants. Previously, a germanium hypersensitive mutant line, KDS-557B, was identified by screening Kitaake mutant rice seedlings with germanium, and a cross between this mutant and the variety Sabine was made for genetic mapping. In FY18, phenotyping of the F2 mapping population was consistent with a recessive trait but results suggested that Sabine may be contributing some tolerance alleles. To simplify the analysis, a cross between KDS-557B and the wild-type progenitor Kitaake was made in FY19 and evaluation of the F1 (i.e. the resulting progeny of the cross) supported the recessive nature of the mutation in KDS-557B. F2 seeds were produced from several F1 plants and an F2 population was grown out in FY20 and preliminary phenotyping experiments confirmed a single gene mutation mode of inheritance. F3 seeds of this population were harvested for additional phenotyping and for generation advance to eventually produce recombinant inbred lines (RILs) through single seed descent. These RILs (F5 or later generation) will facilitate more accurate phenotyping of the germanium hypersensitivity trait. Work on other Kitaake mutants showing altered responses to germanium and screening of a Sabine mutant population for additional germanium tolerance mutants that was delayed in FY19 continues to be postponed due to FY20 maximized telework. In FY19, lack of personnel resources and unusual weather during the spring delayed work on Sub-objective 3B and the government shutdown prevented plans to grow lines in the greenhouse over the winter for field-based evaluations. In FY20, efforts were made to re-establish a nursery at the University of California, Davis rice/wild rice field facility. Due to unexpected increase in university users (including expansion of a project involving weedy red rice that was incompatible with the breeding goals of this sub-objective) and poor field preparation and management, planned experiments could not be established in FY20. This situation was exacerbated by a critical vacancy and maximized telework in FY20. The field facility no longer appears to be viable for this research program and alternative arrangements for FY21 are being identified.

1. New rice grain mutants exhibit altered endosperm resistant to alkali digestion. Gelatinization temperature (GT) is an important trait that affects the cooking and eating quality of rice grains which are of critical importance to consumers. Digestion of milled grains in dilute alkali solution serves as a rapid and inexpensive method to evaluate GT. ARS researchers in Davis, California, have identified two grain mutants with increased tolerance to alkali digestion (i.e. higher GT) and the mutation responsible for one of them. These mutants are valuable genetic resources for studying cooking and eating quality in rice and developing novel rice products for U.S. producers and consumers.

Review Publications
Kim, H., Imatong, R.V., Tai, T. 2020. Identification of rice mutants with altered grain alkali digestion trait. Plant Breeding and Biotechnology. 8:19-27.
Shim, K., Kim, S., Jeon, Y., Lee, H., Adeva, C., Kang, J., Kim, H., Tai, T., Ahn, S. 2020. A RING-type E3 ubiquitin ligase, OsGW2 controls chlorophyll content and dark-induced senescence in rice. International Journal of Molecular Sciences. 21(5).