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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

2019 Annual Report

1a. Objectives (from AD-416):
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.

1b. Approach (from AD-416):
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.

3. Progress Report:
In support of Sub-objective 1A, research continued in determining whether mutations in starch biosynthesis-related genes, which were identified in previous work, have any effect on grain quality. Due to critical vacancies and the extended government shutdown, research efforts were limited to advancing work on characterizing a line harboring a mutation in the transcription factor known as rice starch regulator 1 (Rsr1). This gene effects expression of several starch biosynthesis genes and a previous mutant exhibited altered starch properties and larger grain size. The mutant line NM-5448 exhibits a larger grain size but no significant changes in starch. Two F2 mapping populations derived from crossing the NM-5448 to the wild type progenitor variety Nipponbare and another wild type variety Kitaake in FY18 have been planted this year and will be used to confirm if the large grain phenotype and the mutation in the Rsr1 gene of NM-5448 co-segregate. Filial (F)2 plants with large seed size may be useful for breeding purposes regardless of the genetic basis of this trait. Additional genetic crosses between various grain mutants harboring mutations in starch biosynthesis genes have been made. These include crosses of NM-4936 (a starch branching enzyme I gene mutant characterized and published in FY18) and NM-5448. In support of Sub-objective 1B, evaluation of 13 mutants identified from reverse genetic screening of the silicon/arsenic transport and accumulation genes Lsi1 (three), Lsi2 (nine), and OsABCC1 (one) continued in FY19. Plants from two field trials one in Davis, California, and one in Biggs, California, were harvested and tissue and grain samples are being processed for total silicon and arsenic content analysis. Initial results of total silicon from rice straw samples were generally consistent with FY18 findings which were published this year. Inclusion of a second site (Biggs, California) revealed some differences in silicon content between locations. Analysis of total arsenic in straw and grain samples will be completed in FY19. Backcrossing of some mutants to the wild type progenitor variety Nipponbare was planned for FY19 but delayed until the fall due to critical vacancies and the government shutdown, which has also delayed planting and analysis of progeny from crosses made between mutants in FY18. In support of Sub-objective 2A, grain trait data continues to be collected from F2 mapping populations derived from various genetic crosses of five opaque grain mutants. Because two of the mutants, KDS-1661A and KDS-1852 may be allelic (i.e., mutations affecting the same gene) and due to insufficient human resources in FY19, the primary focus has been on advancing the analysis of the KDS-1830C mutant. Towards that end, additional crosses made between KDS-1830C and wild type Kitaake, the California variety M-103, and another grain quality mutant KDS-1824B (altered alkali spread value/gelatinization temperature) were advanced in FY19 with F1 plants grown and self-fertilized to produce F2 mapping populations which will also be advanced this year for phenotyping at the end of FY19 and beginning of FY20. In FY19, it was observed that some of the F2 progeny from a cross between KDS-2173A and KDS-1852 produced F3 seeds with the white core trait that is typically 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, two to three generations per year. In support of Sub-objective 2B, characterization of these wax crystal-sparse leaf (wsl) mutants including an evaluation of their performance. 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 wild type Kitaake) by wild type Kitaake were planted and individuals exhibiting the wsl (2249D mutant phenotype) were identified and allowed to self-fertilize to produce seeds which will be used for phenotypic evaluations including responses to abiotic and biotic stresses and for preliminary phyllosphere microbiome surveys. Work also continued on the genetic analysis and characterization of the Sabine wsl mutant 6-1A-3, 7-17A-5, 11-39A and 1558.1. In FY19, F2 progeny have been produced by selfing F1 individuals derived from crosses between the mutants (as female parents) and the wild type Sabine (as male parent). Evaluation of the F1 plants was consistent with the previous observations that the mutations responsible for the wsl/wlg (wet leaf glossy) phenotype are recessive. F2 seeds have been produced and will be used for mutation mapping/discovery based on ultrahigh throughput sequencing of phenotypic bulks. Selected F2 individuals will be used to develop advanced backcross lines with the wild type Sabine parent in order to remove background mutations that may confound downstream studies on the effect of these mutations on the response of rice plants to various environmental challenges. Studies by cooperators in Louisiana on the response of some of these wsl mutants 6-1A-3, 7-17A-5, and 11-39A, indicated that they were more susceptible to fall armyworm and rice water weevil than the wild type progenitor variety Sabine. 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 are underway. Wax compositional analysis and quantification of the Sabine wsl mutant 6-1A-3, 7-17A-5, 11-39A and 1558.1 using gas chromatography/mass spectrometry are in progress should be completed by the end of FY19. In support of Sub-objective 3A, a germanium hypersensitive mutant line, KDS-557B, was identified by screening Kitaake mutant rice seedlings with 50 micrometers (uM) germanium, and a cross between this mutant and the variety Sabine was made for genetic mapping. Initial evaluation of a small set of F2 seedlings failed due to difficulties in distinguishing mutant (increased sensitivity as evidenced by more rapid and severe onset of germanium toxicity symptoms) and wild type lines. A dose response test was performed to determine if a lower concentration of germanium would enable more accurate scoring of the mutant phenotype. Evaluation of the response of KDS-557B and wild type varieties, Kitaake, Nipponbare, and Sabine, revealed that five to 15 uM germanium was sufficient to distinguish the mutant from the wild type lines. At five uM, Sabine did not exhibit any symptoms (i.e., lesions due to toxicity) while KDS-557B mutants responded similarly at concentrations from five to 50 uM. 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 variety Kitaake was made and germanium tolerance evaluation of the F1 supported the recessive nature of the mutation in KDS-557B. F2 seeds were produced from several F1 plants and will be used for mutation mapping/discovery in early FY20. Work on other Kitaake mutants showing altered responses to germanium has been delayed due to critical vacancies and the government shutdown. Screening of the Sabine mutant population for additional germanium tolerance mutants has also be delayed to FY20. In regard to Sub-objective 3B, work under this objective has been delayed. Due to continued lack of personnel resources and unusually heavy/prolonged rains during the spring. The government shutdown also prevented plans to grow lines in the greenhouse over the winter for field-based evaluations in FY19. A new management team has been put into place at The University of California, Davis, which has required some adjustment. Plans are in place to re-establish the rice nursery at the University of California, Davis, facility, which should, weather-permitting, facilitate evaluations of lines in FY20.

4. Accomplishments
1. Identification of novel alleles affecting the uptake and accumulation of silicon, arsenic, and germanium in rice. Silicon, arsenic, and germanium are transported from the soil into and within rice plants by transport proteins. While silicon is good for plant growth, arsenic and germanium are toxic and arsenic can cause cancer in humans. An ARS scientist at Davis, California, employed a genetic approach to identify new forms of genes encoding three transport proteins (Lsi1, Lsi2, OsABCC1). Plants with these new genes had altered silicon and arsenic content and sensitivity to germanium. These plants are novel resources for researchers studying transport of these elements and may potentially lead to improved rice varieties for farmers and consumers.

Review Publications
Kim, H., Tai, T. 2019. Identification of novel mutations in genes involved in silicon and arsenic uptake and accumulation in rice. Euphytica. 215:72.
Kim, H., Tai, T. 2019. Identifying a candidate mutation underlying a reduced cuticle wax mutant of rice using targeted exon capture and sequencing. Plant Breeding and Biotechnology. 7(1):1-11.
Yun, Y., Kim, H., Tai, T. 2019. Identification of QTL controlling seedling traits in temperate japonica rice under normal and reduced water availability conditions. Plant Breeding and Biotechnology. 7(2):106-122.