Location: Crops Pathology and Genetics Research
2022 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
In support of Objective 1, research continued on characterizing the effects of novel mutations on grain quality and productivity-related traits in rice. The mutant NM-5448, carries a mutation in the rice starch regulator 1 [rsr1] gene. NM-5448 has larger grains but does not appear to have the changes in grain starch observed in a previously reported rsr1 mutant. The goal of this work is to determine if the rsr1 mutation in NM-5448 is responsible for the larger grain size and to generate molecular tools for use in evaluating starch gene expression and quality-related traits and for breeding. As reported in fiscal year (FY) 2020, two genetic mapping populations were generated by crossing NM-5448 to the varieties Nipponbare (wild-type parent of NM-5448) and Kitaake. Evaluation of the seeds from F2 generation plants of each population was to be completed in FY22; however, the seeds were infested with grain moths which prevented this work. F3 plants of each population were grown in the greenhouse in FY21 and seeds were harvested in FY22. A total of 232 NM-5448/Kitaake F3 plants produced seed; however, the NM-5448/Nipponbare F3 plants were unusually late to flower and very few plants produced seeds. As a result, this generation of the NM-5448/Nipponbare population will need to be re-planted. Double mutant analysis involving genetic crosses of NM-5448 and the mutants NM-4936 and KDS-1830C continues and seeds produced from FY22 plantings will be evaluated in FY23. Data from the elemental analysis of straw samples from 13 rice mutants harboring mutations in the transport and accumulation genes Lsi1 (three), Lsi2 (nine), and OsABCC1 (one) are still being analyzed to determine if the uptake and accumulation of other metalloid elements besides silicon and arsenic are affected. Seeds and straw were harvested from selected mutants grown at a new field site in FY21-22 and are being subjected to analysis to determine if the location where the plants are grown results in different uptake of these elements. In cooperation with researchers at Baton Rouge, Louisiana, the three Lsi1 mutants and their wild-type parent variety, Nipponbare, are being examined for their tolerance to insect pests such as fall armyworm and rice water weevil. Experiments with fall armyworm indicate that larval and pupal weights were higher and developmental times were shorter on the mutants than on Nipponbare. In contrast, experiments with rice water weevils showed no significant difference between the low silicon mutants and Nipponbare regarding the selection of egg-laying sites. Additional seed increases and genetic crosses of the various silicon/arsenic transport and accumulation mutants are being performed this year to provide materials for field-based evaluation of agronomic performance, other biotic stress assays, and for release to gene banks as genetic stocks or breeding germplasm.
In support of Objective 2, research continued on the KDS-1830C grain mutant and an advanced line, 7352-49, identified from a cross between the grain starch mutants KDS-2173A and KDS-1852. In FY22, a total of 179 F3 plants from the cross M-103/KDS-1830C were harvested and the F4 seeds were planted and the resulting F4 plants are being harvested. These F5 seeds will be evaluated to determine if the internal cavity trait in KDS-1830C and in plants derived from backcrossing with the parent variety Kitaake, is also observed in plants derived from crossing with the California variety M-103. The goal of transferring this trait to the M-103 background is to develop germplasm for breeding brewing rice varieties. Generation advance of 7352-49 lines was continued in FY22. The original line K7352-49 had a high proportion of grains with a white-core grain trait that is a characteristic of rice varieties used for brewing premium quality sake (i.e., Japanese rice wine). Using F4 seed of the sibling lines K7352-49.1.1, K7352-49.1.3, and K7352-49.3.2, generation advance was conducted and F5 seeds were harvested and evaluated for the presence and frequency of white-core grains. Seeds from twenty lines (nine, seven and four F5 lines from K7352-49.1.1, K7352-49.1.3, and K7352-49.3.2, respectively) are currently being grown in the greenhouse and the field to evaluate grain and plant traits. Selected plants are being backcrossed to wild-type Kitaake for genetic mapping and germplasm development. To produce enough rice for preliminary brewing evaluations, F5 seeds harvested from a selection of K7352-49.3.2 grown in FY21 were planted in the field this season and will be harvested in the fall for milling and evaluation. Research on the grain mutants KDS-1661A.3 and NE-334.1 and identification of the mutations underlying all the grain mutants has been delayed. Work continued on the genetic analysis and characterization of the cuticle wax-deficient Sabine mutants, specifically mutants 1558.1, 11-39A-1, and 6-1A-3. F2 mapping populations derived from crosses of these mutants with their wild-type parent Sabine were confirmed to be segregating for the wax-deficient trait controlled by recessive single gene mutations. A bulked segregant sequencing approach has been employed using an F2 mapping population from the cross 1558.1/Sabine. Genome sequence data from two DNA pools of twenty F2 plants showing the wax-deficient trait (mutant bulk) and twenty showing the normal wax trait (wild-type bulk) are being analyzed to identify the mutation responsible for the 1558.1 wax-deficient trait. Results of initial evaluation of the response of backcross-derived lines of Sabine mutants 6-1A-3, 7-17A-5, 11-39A-1, and 1558.1 to rice blast fungus suggested that all the wax-deficient mutant lines were more susceptible to the various blast isolates tested by our ARS cooperators in Stuttgart, Arkansas. Seed increases and more genetic crossing to develop lines with fewer potentially confounding background mutations are underway. Results of the stem rot disease testing of the mutant lines provided to cooperators in Biggs, California, in FY21 did not support the highly tolerant rating for 1558.1 in FY19, which may indicate the previous rating was inaccurate or that some other mutation was involved. Additional development of germplasm through crossing and re-testing are needed. In FY22, seed increases of other wax-deficient mutant families (26.1, 264.2, 1086.2, and 2263.1) were completed and M5 generation seed were harvested. One line (2856A.3.3.4) from a previously identified wax-deficient family (2856A.3) was found to have a normal wax trait, indicating that this originally family was either incorrectly identified or during the process of generation advance, the trait was lost. Crosses were made between the wild-type Sabine (male parent) and the mutants. Seeds were recovered from 26.1.2.2/Sabine, 1064.2.1.4/Sabine, and 2856A.3.3.4/Sabine. The wax-deficient mutant crosses will be planted for backcrossing and selfing (i.e., F2 seed production) to map the mutations underlying the 26.1 and 1064.2 mutants. The 2856A.3.3.4 mutant exhibits altered plant morphology compared to Sabine, but further characterization will not be pursued. In addition, an advanced generation line derived from selfing mutant progeny of the cross 6-1A-3/Sabine was backcrossed to Sabine and the resulting progeny, which should have significantly fewer background mutations, will be used to develop lines for further studies on abiotic and biotic stress tolerance. Preliminary surveys of the microbial communities on the leaf surface (i.e., phyllosphere) using a wax-deficient mutant line derived from KDS-2249D, which carries a mutation in the rice wax synthesis gene in the variety Kitaake, have been delayed.
In support of Objective 3, research continued in the screening of rice mutant populations to identify novel mutations that impact agronomic performance, grain quality and reproductive cold tolerance in rice. The work in FY22 involved generation advance of Kitaake mutants in outdoor (448 M7 lines) and greenhouse (136 M7 lines) basins. In addition, a new field site was identified and 263 Kitaake M8 lines were planted in single plots (3 x 3 ft rows with 1 ft. inter-row spacing for each line). A relatively small number of mutant lines were planted due to the new field site and production system, which involves subsurface drip irrigation rather than the traditional rice paddy production system. Trait evaluations are being conducted on the Kitaake M8 lines and trial drone-based multispectral imaging is being performed on a very limited basis in preparation for increased use of this technology. Use of drip irrigation (subsurface and surface) should facilitate evaluation of drought stress response in both the wax-deficient and low silicon mutants as well as screening for additional mutant phenotypes affecting agronomic performance under water-stressed environments. Work on the Sabine (M4 generation) and Nipponbare (M3 generation) populations has been deprioritized due to limited resources. Grow out of M2 seeds from Sabine, Kitaake, and Nipponbare mutant populations for exon capture and sequencing experiments and for M3 seed production continue; however, genetic and physiological characterization of the germanium tolerance mutants has been paused due to additional effort required in setting up the new field site.
Accomplishments
1. Rice mutants uncover differences in insect pest responses to reduced silicon. In rice and other grass species, silicon provides a wide array of benefits, helping to mitigate many environmental stresses. The role silicon plays in the ability of rice plants to defend against important insect pests in U.S. production areas bears further study. ARS researchers in Davis, California, in cooperation with Louisiana State University researchers in Baton Rouge, Louisiana, showed two major insect pests of rice respond differently to low silicon rice mutants. Fall armyworm responded positively to reduced silicon content whereas rice water weevil showed no preference. These mutants are valuable genetic resources for studying the mechanisms through which silicon supports insect tolerance in rice. This knowledge may lead to new strategies for developing insect resistance rice varieties.
Review Publications
Kim, S., Ji, S., Lee, H., Jeon, Y., Shim, K., Adeva, C., Luong, N., Yuan, P., Kim, H., Tai, T., Ahn, S. 2022. A novel embryo phenotype associated with interspecific hybrid weakness in rice is controlled by the MADS-domain transcription factor OsMADS8. Frontiers in Plant Science. 12. Article 778008. https://doi.org/10.3389/fpls.2021.778008.
Kim, D., Kang, J., Shim, K., Kim, H., Tai, T., Ahn, S. 2021. Characterization of genes associated with salt tolerance using transcriptome analysis and quantitative trait loci mapping in rice. Plant Breeding and Biotechnology. 9(4):318-330. https://doi.org/10.9787/PBB.2021.9.4.318.
