Location: Crops Pathology and Genetics Research2018 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:
This new project began in February 2018 and continues research from 2032-21000-021-00D, “Generation and Characterization of Novel Genetic Variation in Rice for the Enhancement of Grain Quality and Agronomic Performance”. See the report for the previous project for additional information. The overall goal is 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. This research builds on previous work involving the generation of rice mutant populations using traditional mutagenesis and the identification of novel gene mutations and mutant phenotypes. 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). Sub-objective 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. The reverse genetics screening for mutations in starch biosynthesis-related genes was completed. Over 150 mutations were identified and confirmed in 14 genes including seven starch synthase genes, four starch branching enzyme genes, two starch debranching enzyme genes, and one regulator of starch biosynthesis gene expression. Preliminary characterization of grain quality traits of mutants is underway. In almost all cases, no visible grain mutant phenotypes have been observed and more detailed analyses (e.g., physics and chemistry tests) are needed to confirm whether the isolated mutations have any effect on gene function. Genetic analysis, including genetic crossing of mutants to develop germplasm for evaluation, is proceeding. Crossing of mutants to generate plants carrying mutations in two different starch genes (e.g., mutations in the genes encoding starch branching enzyme IIa and IIb) has been performed. Double mutants may reveal novel gene interactions and produce new traits. Sub-objective 1B: Characterize the effect of mutations in silicon/arsenic transporters on the uptake and accumulation of these elements in rice. Reverse genetic screening of the silicon/arsenic transport and accumulation genes Lsi1, Lsi2, and OsABCC1 was completed. Twenty-two mutations were detected in total, six in Lsi1, 12 in Lsi2, and four in OsABCC1. Three additional mutations were detected in Lsi1 and OsABCC1 this year. Preliminary evaluation of a subset of 13 mutants (three Lsi1, nine Lsi2, and one OsABCC1) for sensitivity to germanium, a toxic chemical analog of silicon, revealed that one of the Lsi1 mutants (line E-1746) exhibited a mutant phenotype (i.e., no longer sensitive to germanium), similar to published reports of Lsi1 mutants. Preliminary elemental analysis of field-and greenhouse-grown mutants was completed this year and some of the mutants exhibited altered silicon and arsenic content. A second field trial has been planted this year and samples will be analyzed in FY2019. Genetic crossing of some of these mutants to develop lines for more detailed characterization are underway. 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. Sub-objective 2A: Identify the mutations responsible for opaque, non-waxy grain phenotypes and evaluate these mutants for alternative uses. F2 mapping populations from various genetic crosses of five opaque grain mutants (identified previously by visual phenotyping) were planted. DNA has been isolated from four of these populations for mapping in conjunction with grain trait data which is currently being collected. Preliminary results suggest that the grain mutants, KDS-1661A and KDS-1852, may be allelic (i.e., mutations affecting the same gene). The small grain trait of the KDS-1852 was shown to be separate from the opaque grain trait. Evaluation of F2 grains of a cross between the mutant KDS-1830C and the wildtype progenitor line, Kitaake, indicate that the mutant phenotype is controlled by a single gene mutation. Whole-genome resequencing of the KDS-1830C was performed and sequence data are being analyzed to identify candidate gene mutations. An F2 population from a cross of the NE-334 opaque mutant and Kitaake was planted, and preliminary evaluation of plant type and grain suggests that the small plant and grain phenotype of NE-334 are the result of the same gene mutation. The fifth mutant KDS-2173A was previously crossed with the long grain variety L-202. These F2 plants have not yet flowered and will be evaluated in FY2019. Sub-objective 2B: Identify mutations responsible for reduced cuticle wax phenotypes (wax crystal-sparse leaf mutants) and conduct a detailed characterization of these wsl mutants including an evaluation of their performance in the field and response to various biotic stresses. Backcross lines derived from a cross of the wsl mutant KDS-2249D and the wildtype Kitaake were planted and seeds were collected. Backcross lines exhibiting better fertility than the original KDS-2249D mutant line have been identified and will be used for additional evaluation of plant responses to environmental stresses. Additional backcrossing was performed to further purify the KDS-2249D mutation, which resides in the OsGL1-1 gene, from other mutations in the KDS-2249D genetic background. Phenotypic evaluation of F2 mapping populations derived from the crosses of the mutants 7-17A-5, 11-39A, and 1064.2 with a wildtype cuticle wax variety L-202 confirmed that their mutant wsl phenotypes are due to single gene mutations. Previously, crossing of 11-39A and 1064.2 indicated that these mutants are allelic. Crosses were performed between the mutant 6-1A-3 and the variety Kitaake. The resulting F1 plants did not exhibit the wsl phenotype (the mutant was the female parent, thus eliminating the possibility of contamination by self-fertilization). This result indicates that the 6-1A-3 mutation is recessive. Previously, the F1 plants from a cross between 6-1A-3 and the mutant 7-17A-5 were observed to have the wsl phenotype. All the F2 plants from one of those F1 plants exhibited the wsl phenotype as well. Taken together, these data indicate that 6-1A-3 is recessive and allelic to the mutation in 7-17A-5. Additional crosses (including backcrosses to the wildtype progenitor variety Sabine) have been performed to further establish genetic relationships and to develop plants for more detailed trait evaluations. 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. Sub-objective 3A: Identify mutants with altered uptake, transport and accumulation of nutrients, metabolites, and other compounds affecting agronomic performance and grain quality. Previously, 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 wildtype 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 wildtype varieties, Kitaake, Nipponbare, and Sabine, revealed that 5-15 uM germanium was sufficient to distinguish the mutant from the wildtype lines. At 5 uM, Sabine did not exhibit any symptoms (i.e., lesions due to toxicity) while KDS-557B mutants responded similarly at concentrations from 5 to 50 uM. Phenotyping of the F2 mapping population and confirmation of additional putative mutants (i.e., germanium re-screening tests) will be completed this fiscal year and mapping will be performed in FY2019. Sub-objective 3B: Evaluate agronomic and grain quality traits in fixed mutant lines grown under field conditions. Due to lack of personnel resources and management issues at the field site, work under this objective has been delayed. In preparation for FY2019, some lines will be grown over the winter in the greenhouse. In addition, some backcrossing to remove background (i.e., unrelated and undesirable) mutations is ongoing based on previous field observations of some mutant lines in FY2017.
Kim, H., Yoon, M., Chun, A., Tai, T. 2018. Identification of novel mutations in the rice starch branching enzyme I gene via TILLING by sequencing. Euphytica. 214:94. https://doi.org/10.1007/s10681-018-2174-7.