Progress 02/15/19 to 01/19/24
Outputs
Target Audience:Several groups of audience were reached through conferences, seminars, and teaching activities in the past years. We presented the project progress and new discoveries on genetic and molecular mechanisms of seed dormancy, germination and pre-harvest sprouting at five conferences: 1) the 39th Rice Technical Working Group Meeting in Hot Springs, AR, in late Feb of 2023; 2) the project directors' meeting for USDA NIFA AFRI Physiology of Agricultural Plants Program in mid-August of 2023; 3) the 7th International Plant Dormancy Symposium at the University of Western Australia in mid-September of 2023; 4) the 15th International Symposium on Pre-Harvest Sprouting in Cereals in Tsukuba, Japan, in early October of 2023; and 5) the 8th International Hybrid Rice Symposium in Manila, Philippines in mid-October of 2023. The new discoveries were also presented in two seminars at Tasmanian Institute of Agriculture, University of Tasmania, Australia in September, or at the South China Agricultural University, Guangzhou, China, in October 2023. Data from this project were selected as examples or practices in the classes "Molecular Plant Physiology" in the spring semester and "Genome mapping and QTL analysis" in the fall semester of 2023. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two postdoctoral research associates (Drs. Altameemi and Chakraborty) and two graduate research assistants (GRA) at the Ph.D. level (Bhattarai and Xu) were trained with the project in the third year. Dr. Altameemi received training for advanced quantitative genetics to model epistatic interactions using data for the GA signaling and seed dormancy genes. Dr. Chakraborty received training in molecular biology of target genes or gene regulatory networks. Bhattarai continued working on genetic improvement of resistance to pre-harvest sprouting using seed dormancy genes and received training in genomic selection. Xu joined the project in early 2023 and received training in plant genetics, seed biology and RNA-seq data analysis. How have the results been disseminated to communities of interest?Data from this project were presented as both lectures and abstracts in four conferences on rice, seed dormancy, or pre-harvest sprouting. The conferences are: 1) 39th Rice Technical Working Group Meeting in Hot Springs, AR, in Feb of 2023; 2) the 7th International Plant Dormancy Symposium at the University of Western Australia in mid-September of 2023; 3) the 15th International Symposium on Pre-Harvest Sprouting in Cereals in Tsukuba, Japan, in early October of 2023; and 4) the 8th International Hybrid Rice Symposium in Manila, Philippines in mid-October of 2023. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1: To identify interaction partners of SD12a and SD12c in embryos of developing seeds. (100% Accomplished) Final: Several datasets were collected to address questions on Objective 1. Genetic data from isogenic lines grown in the greenhouse and field environments in multiple seasons confirmed that the major QTL qSD12 consists of three underlying genes (SD12a, b and c), each of them has an additive effect on germination inhibition, and two or three of them are cumulative in the effect. Data from a series of molecular biology experiments provided lines of evidence that SD12a and c encode the bHLH family of transcription factors (TFs), the TFs are localized in the nuclear, and both can form homo and hetero dimers. SD12b encodes a protein unknown for molecular function and the molecular data show no interaction of SD12b with SD12a or SD12c. Genomic data for a genomic region of <100 kb show four bHLH domain-containing genes separated by transposable elements. The four genes vary in the length of DNA sequences but are similar in coding sequence. These support the hypothesis that SD12a and c may originate from a DNA sequence in a wild ancestor by two rounds of local duplications and functional differentiation during evolution. Objective 2: To screen direct target genes of SD12a and SD12c for the development of seed dormancy. (100% Accomplished) Final: Three approaches (Y1H, ChIP & RNA-seq) were used to identify target genes of SD12a and/or SD12c, based on the presence of E- and/or G-box binding elements, ChIP DNA sequences, or differentially expressed genes between isogenic lines for allelic variants of the SD12a or c locus. Both SD12a and SD12c can bind the E- and G-box elements in the Y1H assay. Sequencing libraries prepared for the Flag-SD12a and Flag-SD12c transgenic seedlings identified genes containing the E- and/or G-box elements. Many of the E- or G-box-containing genes were differentially expressed in the embryos of 10-d developing seeds between the isogenic lines in RNA-seq analysis. The differentially expressed genes (DEGs) in the 10-d embryos include those encoding heat shock proteins or some other bHLH TFs or involved in the GA biosynthesis/signaling or ABA metabolic pathways. Further research on the GA signaling genes (Slr1, Gid1 and Gid2 and their loss-of-function mutants) identified that all the three genes are involved in the regulation of primary dormancy, and both Slr1 and Gid1 interacted with SD12. The interaction between Slr1 and SD12 was also detected by RNA-seq analysis of genes transcribed in the embryo of germinating seeds. Objective 3: To evaluate individual and combined effects of SD12a, b and c on resistance to pre-harvest sprouting (PHS) in an isogenic background. (100% Accomplished) Final: Effects of qSD12 on delay of germination were evaluated in four types of genetic background, which are an indica-type cultivar, two japonica-type cultivars, a cytoplasmic male sterility (CMS) maintaining line, and a CMS fertility restoration (RF) line. The dormancy-enhancing alleles at the SD12a, b and c loci link in coupling (ABC) in a tropic ecotype of weedy rice and the unbalanced haplotype has not been detected in tested cultivars (abc). So far, we have obtained isogenic lines for five (Abc, aBc, abC, ABc, & AbC) of the six balanced haplotypes, excluding aBC. More than 10 crosses were made to introduce balanced haplotypes into the genetic backgrounds of a CMS maintaining and a CMS RF line. These isogenic lines, together with the CMS maintaining and RF lines, have been genotyped with the 7K SNP Array to facilitate genomic selections across generations of populations from the crosses in the next project. With information, knowledge and plant materials from this project, a four-year collaborative project with a rice breeding program in Arkansas has been initiated to improve the resistance to preharvest sprouting and solve the inadequate germination problem of hybrid (F1) seeds.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2023
Citation:
Tan W, Guo M, Zhu Y, Chakraborty R, Batth BS, Zhang G, Xie D, Gu XY. The Rc and Pb Genes Regulate Seed Pigment Coating, Flavonoids, Dormancy and Germination in Rice.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Gu XY, Bhattarai K, Guo M, Batth BS. Genetic Improvement of Hybrid Rice for Germinability using Linked Genes Controlling Embryo Dormancy. The 8th International Hybrid Rice Symposium Manila, Philippines, Oct. 18-19, 2023
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Yuan Y, Altameemi R, Chakraborty R, Xu H, Gu XY. Involvement of Gibberellin Signaling Pathway in Regulation of Seed Dormancy and Germination in Rice. 15th International Symposium on Pre-Harvest Sprouting in Cereals, Tsukuba, Japan,
Oct 4 -6, 2023.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Gu XY, Bhattarai K, Batth BS, Turnipseed EB. Three Types of Seed Dormancy and Their Evolutionary and Regulatory Mechanisms in Rice. The 7th International Plant Dormancy Symposium, The University of Western Australia
September 11-15, 2023
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Gu XY, Bhattarai K, Guo M, Batth BS. 2023. Genetic improvement of hybrid rice for seed germinability using balanced haplotypes for linked genes controlling embryo dormancy. The 39th Rice Technical Working Group Meeting, Hot Springs, AR. Feb. 20- 23.
|
Progress 02/15/22 to 02/14/23
Outputs
Target Audience:The target audience can be divided into three groups based on events used for dissemination of our results. The first group were attendees at three international or national conferences: 1) The13th Triennial International Society of Seed Science (ISSS) Conference held online on 9-13 August 2021. Attendees included seed biologists and technologists from universities, institutes, or seed companies. We delivered an abstract and an oral presentation to the ISSS conference. 2) The XXIX Plant and Animal Genome (PAG) Conference held online on 7-12 January 2022. Attendees included scientists and researchers working areas of genetics, genomics, molecular biology, or agriculture. We delivered two abstracts and two e-posters to the PAG conference. 3) The annual meeting of multi-state project for seed biology held at Cornell AgriTech, Geneva, NY in mid-October 2022. Attendees included seed biologists, technologists, and agronomists. We delivered an oral presentation to the meeting. The second audience included farmers or agronomists who participated in the Field Day organized by Missouri Rice Council in late August of 2021. We presented two posters on the Missouri Rice Research Farm and gave two online oral presentations during the field day. The third group included graduate students in the PD's classes PS792 and BIOL664. PS792 was "Genome Mapping and QTL Analysis" opened for 9 attendees in the fall semester of 2021. BIOL664 was "Molecular plant Physiology" opened for 7 graduate students in the spring semester of 2021. The PD carefully selected histological, molecular biological, genomic, and quantitative genetic data from the rice project as examples for lectures or for real data practices. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two postdoctoral research associates (PRA) and two PhD graduate research assistants (GRA) were trained with the above-stated experiments. The PRA Rami Altameemi worked on experiments to identify interactions between the GA and SD genes and received extensive training for large-scale marker genotyping, QTL mapping, and marker-assisted selections. The PRA Rupak Chakraborty focused on transcriptomic analysis by RNA-sequencing and received training in seed biology, plant genetics, gene expression analysis, and annotation, and advising graduate students. PhD student Kamal Bhattarai worked on genetic experiments on SD12s in the greenhouse and field environments and received training in seed biology, marker genotyping, linkage mapping, and marker-assisted selection. The new PhD student Marya Bibi was involved in some genetic or molecular biological experiments and received training for quantitative genetics, seed biology, and marker genotyping. How have the results been disseminated to communities of interest?Some of the experimental results were presented in the Seed Biology Multi-State Project Annual Meeting in Tucson, AR in October 2022, or submitted to the Rice Technical Working Group Meeting in Hot Springs, AR, in late February 2023. Some of the data from the project were used as real data for the PD's teaching for "Crop Physiology" (PS792) or for "Molecular Plant Physiology" (BIOL/PS 664). What do you plan to do during the next reporting period to accomplish the goals?Objective #1: To identify interaction partners of SD12a and D12c in embryos of developing seeds. Complete analyses of RNA-sequencing data for objective 2 and select candidate genes to confirm their functions for the development or release of seed dormancy. Keep advancing the introduction of the SD12 gene(s) into genetic backgrounds of commercial MS or RF varieties by marker-assisted backcrossing strategies. Objective #2: To screen direct target genes of SD12a and D12c for the development of seed dormancy. Confirm functions of candidate genes detected by the RNA-seq analysis for their functions on the development of embryo dormancy using molecular biology techniques. Objective #3: To evaluate individual and combined effects of SD12a, b and c on resistance to pre-harvest sprouting (PHS) in an isogenic background. Advance the F2, F3, or backcross populations to evaluate effects of individual SD12s on seed dormancy and resistance to pre-harvest sprouting. Develop collaborations with rice breeders in AR and LA to extend the research to breeding programs.
Impacts
What was accomplished under these goals?
Objective 1: To identify interaction partners of SD12a and SD12c in embryos of developing seeds. (100% Accomplished) Map-based cloning of qSD12 identified three (SD12a, b, and c) underlying genes linked on a short (70 kb) genomic region. SD12b is unknown for molecular function, while SD12a and SD12c are annotated as the bHLH family transcription factors (TFs), based on DNA and deduced protein sequences. A bHLH TF is expected to form a dimer to regulate expression of downstream genes in the nuclei. A series of experiments, including yeast-two-hybridization, bimolecular fluorescence complementation, and immunoprecipitation assessments provided evidence that the SD12a and SD12c proteins locate in the nuclei, each of them could form homodimers (SD12a-SD12a and SD12c-SD12c), and both could form a heterodimer (SD12a-SD12c). These results imply: 1) SD12a and SD12c could work independently in different genotypes (lines) or work together in the same genotype to regulate expression of genes for the development or release of seed dormancy; 2) SD12a or SD12c could also interact with the other bHLH TFs (e.g., those detected by transcriptomic analyses for the second objective) to influence seed dormancy and germination; and 3) allelic variants isolated from the SD12a, SD12c, or both loci can be used to manipulate cultivars for the level of primary seed dormancy and germinability in crop breeding. The SD12a, SD12b, and SD12c genes link in coupling to increase seed dormancy in the donor parent of weedy rice. The linkage makes it difficult to prove the molecular interactions detected in the above-stated experiments. This problem can be overcome by use of the SD12a, SD12b, and SD12c transgenes, which were randomly inserted into the genome of a non-dormant line. Thus, F2 populations from crosses between transgenic T5 lines for SD12a, SD12b, or SD12c were developed and the F2 plants are being evaluated for the degree of seed dormancy by germination testing. Data from the populations will be used to model epistatic interactions. Information from the modeling is of both agricultural and fundamental importance, in terms of crop breeding to manipulate germination ability of inbred or hybrid varieties and underlying of evolutionary mechanisms for the dormancy trait and for a multigenic QTL. Objective 2: To screen direct target genes of SD12a and SD12c for the development of seed dormancy. (95% Accomplished) Both transcriptomic and genetic approaches have been conducted to identify genes regulated by the transcription factor genes SD12a and SD12c. The transcriptomic analysis was first focused on genes expressed in the embryo of developing seeds from the isogenic lines for SD12a, SD12b, SD12c, or their combination. Candidate genes identified from the analysis include those involved in the gibberellin (GA) biosynthesis or signaling pathways. The GA signaling pathway consists of three core factors/genes: SLR1 encoding the DELLA protein, GID1 encoding the GA receptor, and GID2 encoding the F-box protein in rice. Thus, genetic experiments were conducted to evaluate allelic differences of the Slr1/slr1, Gid1/gid1, or Gid1/gid2 loci in germinability. Three seed populations (1000 seeds/population) segregating for each of the three loci were genotyped for the GA genes and evaluated for time of imbibition to germination in a controlled environment. The experimental results revealed that the Slr1 functional allele delays germination, while the Gid1 or Gid2 functional alleles promote germination. Based on these datasets, we hypothesized that the three genes may work as a unit to control seed dormancy release and germination. Two new experiments are being conducted to prove the hypothesis. One of the new experiments was to combine allelic variants for both GA (Slr1/slr1, Gid1/gid1, or Gid1/gid2) and seed dormancy (SD) loci in the same hybrids and hybrid F2 populations. The three F2 populations were developed in a greenhouse and seeds harvested from individual plants. Marker genotyping and seed dormancy assessment have been completed for the population involving the Slr/slr1 alleles. Association analysis between the genotyping and phenotypic data revealed epistatic interactions between the Slr1 locus and SD12 or SD7-1. Experiments on the other two populations involving the Gid1 or Gid2 locus are on-going. The other new experiment was a transcriptomic analysis to identify genes regulated by Slr1 to inhibit or promote germination. Total RNAs were prepared from three genotypes of embryos (Slr1Slr1, Slr1slr1, and slr1slr1) in germinating seeds and sequenced. RNA-sequencing analysis revealed that genes regulated by Slr1 include: 1) natural genes for seed dormancy, such as SD12a and the orthologue of the Arabidopsis DOG1 gene; 2) predicted genes for the ABA signaling; and 3) others for hydrolyses required for the cell wall expensing and mobilization of seed nutritive reserves. These sets of information are new to the research direction and prompt us and other groups to fill knowledge gaps between seed dormancy and the GA signaling pathway in near future. Objective 3: To evaluate individual and combined effects of SD12a, b and c on resistance to pre-harvest sprouting (PHS) in an isogenic background. (90% Accomplished) A total of 80 F4 lines from four crosses with a male-sterility (MS) line and 40 F3 lines from two crosses with a fertility-restoration (RF) line were evaluated for agronomic traits in a summer field experiment at the Arkansas Rice Research and Extension Center. The size for each of the 120 lines was 60 to 100 plants to select genotypes that not only contain the dormancy-enhancing alleles at one or more of the SD12a, SD12b, and SD12c loci, but also have excellent agronomic traits (plant morphologies, flowering time, and yield components). Twenty of the F4 lines were genotyped for SD12s and evaluated for seed dormancy by germination testing, and the data revealed effects of SD12s on the duration of primary dormancy and resistance to pre-harvest sprouting. These and some other lines are being used as genetic materials for a dissertation project of newly recruited graduate student.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2023
Citation:
Gu XY, Bhattarai K, Guo M, Batth BS. 2023. Genetic improvement of hybrid rice for seed germinability using balanced haplotypes for linked genes controlling embryo dormancy. The 39th Rice Technical Working Group Meeting, Hot Springs, AR. Feb. 20- 23.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Gu X. 2022. Research progress on seed dormancy, longevity, and germination in rice. Annual Meeting of Seed Biology Multistate Research Project (W4168). Tucson AZ, Oct. 14.
|
Progress 02/15/21 to 02/14/22
Outputs
Target Audience:The target audience can be divided into three groups based on events used for dissemination of our results. The first group were attendees at three international or national conferences: 1) The13th Triennial International Society of Seed Science (ISSS) Conference held online on 9-13 August 2021. Attendees included seed biologists or technologists from universities, institutes, or seed companies. We delivered an abstract and an oral presentation to the ISSS conference. 2) The XXIX Plant and Animal Genome (PAG) Conference held online on 7-12 January 2022. Attendees included scientists or researchers working areas of genetics, genomics, molecular biology, or agricultures. We delivered two abstracts and two e-posters to the PAG conference. 3) The annual meeting of multi-state project for seed biology held at Cornell AgriTech, Geneva, NY in mid-October. Attendees included seed biologists, technologists, or agronomists. We delivered an oral presentation to the meeting. The second audience included farmers or agronomists who participated in the Field Day organized by Missouri Rice Council in late August of 2021. We presented two posters on the Missouri Rice Research Farm and gave two online oral presentations during the field day. The third group included graduate students in the PD's classes PS792 and BIOL664. PS792 was "Genome Mapping & QTL Analysis" opened for 9 attendees in the fall semester of 2021. BIOL664 was "Molecular plant Physiology" opened for 7 graduate students in the spring semester of 2021. The PD carefully selected histological, molecular biological, genomic, or quantitative genetic data from the rice project as examples for lectures or for real data practices. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided opportunities to train three doctoral level students, Bhupinder Batth, Ahmed Charif, and Kamal Bhattarai during the reported period. Batth was continuously trained for experimental designs, data collection, and data analyses for molecular biology, plant genetics, seed biology and marker-assisted selection for seed-related traits. Charif was continuously trained for ecological genomics, high-throughput genotyping and modeling G-by-E interactions using the genotyping data. Bhattarai, a newly recruited graduate research assistant for this project, was trained for genetics and seed biology, such as hybridization with isogenic lines for specific genes, genotyping and phenotyping of a population to identify CRISPR/Cas9-induce mutants, and seed dormancy assessment by standard germination testing. In addition, two postdoctoral associates (Drs. Min Guo and Yuan Yaun), a visiting scientist (Dr. Hang Yu), and a part-time research assistant (Rami Altameemi, a Ph.D. candidate) were trained during this period. Dr. Guo was trained for gene expression analysis, recurrent backcrossing with marker-assisted selection, seed accelerated aging, and modeling epistatic and G-by-E interactions. Dr. Yuan was trained for detecting temporal and spatial expression patterns of seed dormancy or GA signaling genes using both marker-assisted genetic and transcriptomic strategies. Dr. Yu was continuously trained for transcriptomic analysis of embryo dormancy using RNA-sequencing, including modeling the temporal expression patterns of genes regulated by SD12s. Mr. Altameemi in the last semester of his Ph.D. program in crop breeding was trained for seed biology and basics of molecular biology such as DNA extraction and quantification, PCR, and electrophoresis. How have the results been disseminated to communities of interest?Some of the results from this period were released in a book chapter and a peer-review paper, or as posters and oral presentations on a Rice Field Day in Missouri and three national/international conferences. They are the 13th International Society of Seed Science conference, the 2021 Annual meeting for the multi-state project of seed biology, or the XXIX Plant and Animal Genome conference. What do you plan to do during the next reporting period to accomplish the goals?Objective #1: To identify interaction partners of SD12a and D12c in embryos of developing seeds. Confirm the molecular interaction between the SD12a and SD12c transcription factor genes and evaluate the interactional effects on seed dormancy using the transgenes cloned from a donor of weedy rice. Objective #2: To screen direct target genes of SD12a and D12c for the development of seed dormancy. Confirm functions of candidate genes detected by the RNA-seq analysis for their functions on the development of embryo dormancy using molecular biology techniques. Objective #3: To evaluate individual and combined effects of SD12a, b and c on resistance to pre-harvest sprouting (PHS) in an isogenic background. Advance the F2, F3 or backcross populations to evaluate effects of individual SD12s on seed dormancy and resistance to pre-harvest sprouting. Develop collaborations with rice breeders in AR and LA to extend the research to breeding programs.
Impacts
What was accomplished under these goals?
developing seeds. (90% Accomplished) Hybrid F1s between T5 lines for the SD12a and SD12c transgenes from a line of weedy rice were developed to confirm the molecular interaction between the two bHLH transcription factor genes linked in coupling. Objective 2: To screen direct target genes of SD12a and SD12c for the development of seed dormancy. (90% Accomplished) The DEGs detected by RNA-seq analysis were annotated for temporal expression patterns in the embryo tissue during seed development and for gene regulatory networks (GRNs). There were basically two contrasting patterns of temporal expression in 7- to 42-d seeds between the up- and down-regulated DEGs by SD12a, SD12b, SD12c or their combination (SD12a, b & c). Putative GRNs were developed for the SD12a and SD12c bHLH transcription factors based on the G and/or E DNA-binding boxes and molecular functions, such as the genes involved in the GA or ABA singling pathways or encoding heat shock or late embryogenesis abundant proteins for resistance to abiotic stress during seed maturation programs. Objective 3: To evaluate individual and combined effects of SD12a, b and c on resistance to pre-harvest sprouting (PHS) in an isogenic background. (80% Accomplished) Statically significant effects of SD12a, b, c, or their combinations on the degree of seed dormancy were detected in genetic backgrounds of an indica or a japonica cultivar, based on germination data collected from the F2 populations grown in a field environment. However, the effect size varied with the genetic backgrounds, which will be confirmed with advanced generations of the populations in a greenhouse environment. Marker-assisted backcrosses were advanced to the BC2F1 generation for SD12a, b and c in the genetic background of a male sterility maintaining line or the BC1F1 generation for SD12a in the background of a fertility restoration line.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2022
Citation:
Gu X-Y. 2022. Seed dormancy genes and their associated adaptive traits underlie weed persistence: A case study of weedy rice. In: Persistence Strategies of Weeds in Agriculture edited by Upadhyaya, M.K., Clements D.R. and Shrestha A. Wiley, NY.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Liu T, Sun L, Meng Q, Yu J, Weng L, Li J, Deng L, Zhu Q, Gu X-Y. Chen C, Teng S, Xiao G. 2021. Phenotypic and genetic dissection of cadmium accumulation in roots, nodes and grains of rice hybrids. Plant Soil 463: 3953 (published on March 01).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Gu X-Y, Yuan Y, Hu Q. 2021. Involvement of gibberellin signaling pathway in regulation of seed dormancy and germination in rice. 13th Triennial Meeting of International Society for Seed Science. Hosted Online by The Royal Botanic Garden, Kew. August 9-13. (Oral presentation and abstract)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Gu XY. 2021. Research progress in regulatory mechanisms of seed dormancy and longevity in rice. The 2121 Annual Meeting for the Multi-state Project of Seed Biology. Cornell Agr-Tech Center. Geneva, NY. Oct. 14 16.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Yu H, Feng J, Li Y, Gu X-Y. 2022. Transcriptomic analysis of embryo dormancy development regulated by SD12s in rice. The XXIX Plant and Animal Genome Conference. San Diego, CA. Jan. 7-12.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Charif A, Wang J, Batth BS, Turnipseed EB, Gu X-Y. 2022.
Ecological genomics of seedbank longevity in weedy rice.
The XXIX Plant and Animal Genome Conference. San Diego, CA. Jan. 7-12.
|
Progress 02/15/20 to 02/14/21
Outputs
Target Audience:There were two groups of interested parties for this research who were reached in year 2. One group was the rice research and extension community of approximately 200 who met at the Rice Technical Working Group Conference in late February 2020. Two abstracts developed from this continuous research were selected as oral presentations. One was by the breeding, genetics, and genomics program (Pyramiding of genes controlling seed dormancy or longevity to improve rice varieties for resistance to pre-harvest sprouting) and the other was by the weed control and regulation program (Effects of seed dormancy genes on soil seed bank longevity of weedy rice in till and no-till cropping systems). The other group was students in the classes "BIOL664 Molecular plant Physiology" in the spring semester of 2020 (12 graduate students) and "PS763 Crop Physiology" in the fall semester of 2020 (15 graduate students). The PD selected genomic or seed biology data from our research as examples of real data for use in the classes. Changes/Problems:Both genetic and molecular (particularly the ChIP analysis) experiments were negatively impacted due to the COVID-19 pandemic. The ChIP experiments in the collaborator's lab have been challenged because of difficulty in the enrichment of DNA sequences from transgenic lines for cDNAs of SD12a, b or c. The PD may apply for an extension of the project near the end of the third year. What opportunities for training and professional development has the project provided?Three graduate students [Yeter Karakoc (MS), Bhupinder Batth and Ahmed Charif (PH.D.)], two postdoctoral researchers (Drs. Min Guo and Yuan Yuan), and a visiting scientist (Hang Yu, a Ph.D. candidate) were trained with this project. Karakoc was trained for basics of plant molecular biology and QTL analysis, competed her thesis titled "Reverse genetic analysis of the Ehd1 gene for association between seed dormancy and flowering time in rice" and received her Master of Science degree. Batth was trained for molecular biological techniques, developing hybrids and segregating population, seed biology and techniques, and CRISPR/Cas9-induced mutagenesis in rice. Charif was trained for high-throughput genotyping with an SNP array platform and analysis of the genotyping data, including constructing a high-resolution of genetic map and QTL mapping. Dr. Guo was trained for marker-assisted backcrossing to improve crop resistance to pre-harvest sprouting, genetic analysis of seed dormancy genes for epistatic, and G-by-E interactions and advising graduate students. Dr. Yuan was trained for mutant analyses of the GA signaling genes, molecular techniques to characterize genes for GA signaling and seed dormancy, and genotyping embryos from large populations of seeds segregating for each of the GA genes. Yu was trained for transcriptomic analysis using RNA-sequencing, bioinformatics to annotate differentially expressed genes for the development of embryo dormancy, and CRISPR/Cas9-induced mutagenesis in rice. How have the results been disseminated to communities of interest?The results were released as publications in one journal article (Molecular Breeding), one book chapter (Chapter 4 in Persistence Strategies of Weeds in Agriculture), two abstracts in the symposium of "2020 Rice Technical Working Group Conference", and a thesis. Some of the results were released in two oral presentations to the "2020 Rice Technical Working Group Conference." Some of the results and genetic materials were communicated with rice breeder and researchers working on the Missouri Rice Research Farm, and rice farmers in Southeast Missouri. Some of genetic, molecular, or seed biological data were selected as examples in the PD's teaching for the classes "Molecular Plant Physiology" or "Crop Physiology". What do you plan to do during the next reporting period to accomplish the goals?Objective #1: To identify interaction partners of SD12a and D12c in embryos of developing seeds. Evaluate the three digenic (Ab//aB, Ac/aC, and Bc//bC) heterozygotes of s for international effects on gene expression and the degree of seed dormancy. Objective #2: To screen direct target genes of SD12a and D12c for the development of seed dormancy. Annotate differentially expressed genes (DEGs) identified from 10-d embryos of the five isogenic lines using databases for rice, other cereal crops, or model plants (e.g. Arabidopsis thaliana); determine expression profiles of the DEGs during the seed maturation period to infer their functions for the development of seed or embryo dormancy; and characterize the GA signaling genes (Slr1/srl1, Gid1/gid1 & Gid2/gid2) for transcription abundance and effects on germination ability in the background where all the SD12 alleles are functional for seed dormancy. Objective #3: To evaluate individual and combined effects of SD12a, b and c on resistance to pre-harvest sprouting (PHS) in an isogenic background. Complete marker genotyping and phenotypic identification of seed dormancy for the F2 populations obtained in late 2020 to estimate additive and dominance effects of seed dormancy gene(s) of SD12s in different genetic backgrounds; Keep advancing backcrossing (BC1F1 or BC2F1) populations to introduce one or more of the dormancy-enhancing alleles into the genetic background of the male sterility maintaining line of hybrid rice; and select homozygous genotypes of elite agronomic traits from the F2 or BC2F2 populations as germplasms for distributions to breeders.
Impacts
What was accomplished under these goals?
Objective #1: To identify interaction partners of SD12a and D12c in embryos of developing seeds. (80% Accomplished) Protein-protein interaction between SD12a and SD12c was confirmed by biochemical (BiFC and Y2H) experiments. Both BiFC and Y2H assessments failed to detect an interaction of SD12b with SD12a or SD12c. Natural variants of the , and loci in our experiment system are linked in coupling, such as . The three wild-type alleles on the ABC haplotype of <100 kilo bases from the donor of weedy rice were genetically dissected from each other. The three dissected alleles , and in isogenic lines were used to develop digenic hybrids in the heterozygous genotypes , and , in which the two loci are linked in repulsion. The three digenic heterozygotes will be used to determine international effects of the two loci on transcription abundance and the degree of seed dormancy. Objective #2: To screen direct target genes of SD12a and D12c for the development of seed dormancy. (70% Accomplished) The embryos isolated from 10-d developing seeds of the isogenic lines were able to germinate on MS medium with 2% sucrose. The transcripts of SD12a, SD12b, and SD12c reached the peak level at 10 d after flowering. These two lines of information imply that the embryo dormancy controlled by SD12s is developed after embryogenesis or during the seed maturation period. RNA-sequencing analysis identified 23.3-24.9k genes expressed in the 10-d embryos from five isogenic lines (abc, Abc, aBc, abC, and ABC). The number of differentially expressed genes (DEGs) between the isogenic lines varied from 450 to 550 for single genes (i.e., SD12a, b or c) to 1,200 to 2,000 for two or all of the three genes. A set of new molecular genetic experiments revealed, likely for the first time, that the gibberellin (GA) signaling genes , and are also involved, not only in regulation of germination velocity, but also in the development of embryo dormancy. Transcriptional profiles of the GA signaling genes in germinating embryos were developed, and genic (both additive and dominance) effects of the three loci on the degree of embryo dormancy were evaluated in the genetic backgrounds of the cultivars Nipponbare or Taichong 65, which are natural mutants for the , and loci. Objective #3: To evaluate individual and combined effects of SD12a, b and c on resistance to pre-harvest sprouting (PHS) in an isogenic background. (60% Accomplished) A set of genetic experiments identified strong effects of genotype-by-environment (G-by-E) interactions on the degree of primary dormancy. The experiments were conducted using 16 isogenic lines for tetra-genic recombinants of the SD1-2, 7-1, 7-2 and 12 loci under both greenhouse and field conditions. The trigenic QTL SD12 had the largest genetic effect on germination inhibition in both environments, but the effect size was smaller in the field than in the greenhouse due to the G-by-E interaction. About 10 hybrid populations derived from crosses between each of the SD12A, SD12B, SD12C, SD12AB, and SD12ABC isogenic lines and a male sterility maintaining line, or a ClearField cultivar were advanced to the F2 generations. The plant populations were grown in the Missouri Rice Research Farm or in greenhouses. Marker genotyping and seed dormancy assessment have been done for about a half of the F2 populations. Marker-assisted backcrosses to introduce one or more dormancy-enhancing alleles from the isogenic lines into the male sterility maintaining line were advanced to the BC2F1 generation.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Wang, J., Korkmaz, U., Guo, M., Pipatpongpinyo, W., Gu, X.-Y. 2020. Pyramiding seed dormancy genes to improve resistance of semi-dwarf varieties to pre-harvest sprouting in rice. Molecular Breeding. 40, 93. https://doi.org/10.1007/s11032-020-01172-2.
- Type:
Journal Articles
Status:
Other
Year Published:
2021
Citation:
Gu X-Y. 2021. Seed dormancy genes and their associated adaptive traits underlie weed persistence: A case study of weedy rice. In: Persistence Strategies of Weeds in Agriculture edited by Upadhyaya, M.K., Clements D.R. and Shrestha A. Wiley, NY (In Press).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Gu, X.-Y., Wang, J., Korkmaz, U., Charif, A., Guo, M. 2020. Pyramiding of seed dormancy and longevity genes to improve resistance to pre-harvest sprouting and germinability of rice. The 38th Annual Rice Technical Working Group Conference. Orange Beach, AL. Feb. 24-27.
- Type:
Theses/Dissertations
Status:
Submitted
Year Published:
2020
Citation:
Karakoc, Y.S. 2020. Reverse genetic analysis of Ehd1 for the association between seed dormancy and flowering time in rice. M.S. Thesis, South Dakota State University, Brookings, SD.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Pipatpongpinyo W, Korkmaz U, Wu H, Kena A, Ye H, Feng J, Gu X-Y. 2020. Assembling seed dormancy genes into a system identified their effects on seedbank longevity in weedy rice. Heredity. 124:135-145.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Gu X-Y, Feng J.A. 2019. Transgenerational pattern of epigenetic inheritance for seed dormancy associated with qSD12 in rice. The 6th Workshop on Molecular Aspects of Seed Dormancy and Germination. Volendam, The Netherlands. September 22-25. (Abstract #31).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Karakoc Y, Wang J, Pipatpongpinyo W, Gu X-Y. 2019. Reverse genetic analysis of the Ehd1 gene for the association between seed dormancy and flowering time in rice (Oryza sativa L.). The 2019 ASA-CSSA-SSSA International Annual Meeting. San Antonio, TX. Nov. 10-13.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Karakoc Y, Wang J, Gu X-Y. 2019. Mapping of Ehd1-regulated seed dormancy and flowering time genes in rice (Oryza sativa L.). The 12th Annual Plant Science Symposium. University of Minnesota, Saint Paul, MN. March 28-29.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Karakoc Y, Wang J, Gu X-Y. 2019. Mapping of quantitative trait loci associated with seed dormancy and flowering time in rice (Oryza sativa L.). The 35th Annual Plant Science Graduate Student Symposium. North Dakota State University, Fargo, ND. March 15-16.
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2019
Citation:
Luai M. 2019. A seed dormancy-mediated biotechnology to mitigate risk of transgene flow into weedy rice. Dissertation. South Dakota State University, Brookings, SD.
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Progress 02/15/19 to 02/14/20
Outputs
Target Audience:The target audiences reached during the past reporting year included: 1) Seed biologists and researchers at the annual meeting of the USDA multi-state project W4168: Environmental and Genetic Determinants of Seed Quality and Performance, in Lexington, KY from October 10-12, 2019; 2) Seed biologists/researchers, technologists, and breeders from universities, institutes, and seed industries at the 6th International Symposium on Molecular Aspects of Seed Dormancy and Germination in Volendam, The Netherlands from September 22-25, 2019; 3) Graduate students in theclass "PS792, Genome Mapping and QTL Analysis" at South Dakota State University in the fall semester of 2019; and 4) Graduate students in the class "BIOL664, Molecular Plant Physiology" at South Dakota State University in the spring semester of 2019. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project trained one postdoctoral associate (Dr. Luis Vaschetto) and three visiting scholars (Drs. Junwei Wang, Wenwu Tang and Min Guo) in the areas of genetics/epigenetics, molecular biology, and seed biology. Dr. Vaschetto was recruited specifically for this project started in June 2019. Drs. Wang (2-11/2019) and Tang (2-9/2019) were visiting scholars who worked part-time on this project. Dr. Guo has been a part-time postdoctoral research assistant for this project since mid-June 2019. All these postdoctoral fellows participated in writing review/research papers or research proposals for topics of genetic, epigenetics, or molecular mechanisms of seed dormancy or resistance to pre-harvest sprouting in cereal crops. The project also provided training for three graduate research assistants (Luai Muhammad, PhD; Yeter Karakoc, MS; and Bhupinder Singe Batth, PhD) in gene mapping, cloning, sequencing or functional analysis by RNAi, and seed biology. Muhammad's dissertation project focused on silencing seed dormancy genes including SD12, and this project concluded in December of 2019. Karakoc's thesis project (2018-2020) focused on reverse genetic analysis of seed dormancy genes in rice. Batth's dissertation project was initiated in September of 2019 and his current focus is on genetic effects of seed dormancy genes, including SD12s, in the genetic background of ClearField® rice. These graduates participated in paper/proposal writing. How have the results been disseminated to communities of interest?Results from this project were presented at two international conferences: The 6th International Symposium on Molecular Aspects of Seed Dormancy and Germination in Volendam, The Netherlands from September 22-25, 2019 and The 2019 ASA-CSSA-SSSA International Annual Meeting, San Antonio, TX, Nov. 10-13, 2019. Results were also presented at three national/regional conferences: The annual meeting of USDA multi-state project W4168: Environmental and Genetic Determinants of Seed Quality and Performance in Lexington, KY from October 10-12, 2019, The 12th Annual Plant Science Symposium at University of Minnesota, Saint Paul, MN, March 28-29, 2019, and The 35th Annual Plant Science Graduate Student Symposium, North Dakota State University, Fargo, ND, March 15-16, 2019. Experimental data from this project were used as examples or as real data for practices in theclasses "PS792, Genome Mapping and QTL Analysis" in the fall semester of 2019, and "BIOL664, Molecular Plant Physiology" at South Dakota State University in the spring semester of 2019. What do you plan to do during the next reporting period to accomplish the goals?Objective #1: To identify interaction partners of SD12a and D12c in embryos of developing seeds. In year 2 we will screen for putative partners of SD12a and SD12c using the established Y2H system. We will also confirm the candidate partners using the qRT-PCR, BiFC or ChIP systems. Objective #2: To screen direct target genes of SD12a and D12c for the development of seed dormancy. In year 2 we will use transcriptomic analysis to identify differentially expressed genes (DEGs). This includes: 1) planting a set of isogenic lines for a set of allelic variants at SD12a, SD12c or both under controlled conditions to prepare high quality RNA samples from the embryo tissue of developing seeds; and 2) identifying DEGs using RNA-seq analysis. Co-IP and ChIP-seq analyses of candidate genes will also be conducted. Transgenic lines for Flag-SD12a and Flag-SD12c have been developed in the collaborator's lab. On-going experiments include developing Illumina sequencing libraries and sequencing ChIP DNA segments. Objective #3: To evaluate individual and combined effects of SD12a, b and c on resistance to pre-harvest sprouting (PHS) in an isogenic background. Hybrid F1 plants will be grown in a greenhouse. Recurrent backcrossing with marker-assisted selection will be used to transfer one or more of the SD12s into the male sterility or restoration lines. Seed dormancy will be evaluated the hybrid F1 and parental plants. A mist system will be installed and used to evaluate genotypic difference in the resistance to pre-harvest sprouting.
Impacts
What was accomplished under these goals?
Objective #1: To identify interaction partners of SD12a and D12c in embryos of developing seeds. (60% Accomplished) SD12a and SD12c are two of the three tightly linked genes underlying , a quantitative trait locus (QTL) with the largest effect on seed dormancy in rice. Genomic DNAs and full-length cDNAs of SD12a and SD12c were sequenced, and the sequences annotated with different genomic tools and protein databases. The annotations revealed that allelic variants at SD12a and SD12c from some lines of weedy/wild rice are functional, and encode the bHLH family transcription factors (SD12a and SD12c). Both SD12a and SD12b are located in nuclei, as determined by red and green fluorescence protein (R/GFP) assessments, suggesting that they have a potential to function as regulatory proteins controlling gene expression or transcription. Functional bHLH family transcription factors (TFs) require two units (partners) of either identical or different proteins, known as homodimers or heterodimers. Data from a series of experiments revealed that each of SD12a and SD12c can form homodimers, and can also interact with each other to form a heterodimer in a bimolecular fluorescence complementation (BiFC) system. These results were also confirmed by a set of yeast-two-hybridization (Y2H) assessments. More genomic DNA or cDNA sequences for and were generated from this project or collected from recent reports. Phylogenetic analysis of the sequences indicate that and are closely related in protein structure and function, and they may originate from local duplications of the same ancestor gene during evolution. The above-stated experimental systems are being used to identify additional partners interacting with and/or Little is known about molecular mechanisms that regulate the development of embryo dormancy to block germination. The above results provided evidence that natural variation in embryo dormancy can be controlled at the transcriptional level, and the SD12a and SD12c transcription factors are able to work independently or interdependently to regulate expression of genes important for the development of embryo dormancy. In addition to the following research in this project, the above information is also important for research on evolutionary mechanisms of seed dormancy, a key adaptive trait for flowering plants. Objective #2: To screen direct target genes of SD12a and D12c for the development of seed dormancy. (20% Accomplished) The bHLH familiar transcription factors regulate gene expression by recognizing short DNA-binding sequences (motifs) in promoter regions of downstream genes, including the E (CANNTG) and G (CACGTG) motifs. Yeast-one-hybridization (1H) experiments were conducted for both SD12a is able to bind the E-box and the G-box, while SD12c is able to bind with the E-box element only, in vitro. Experimental systems were developed to identify genes regulated by SD12a and SD12c using regular RNA-seq analysis and chromatin immunoprecipitation (ChIP) sequencing analysis. The systems include a set of isogenic lines for allelic variants at each of the and loci, and transgenic lines for each of the flag-tagged and . Objective #3: To evaluate individual and combined effects of SD12a, b and c on resistance to pre-harvest sprouting (PHS) in an isogenic background. (30% Accomplished) Lack of seed dormancy occasionally causes pre-harvest sprouting, a worldwide problem in cereal crop production. Allelic variants for each of the , , , + were isolated into an isogenic background. These isogenic lines were evaluated for seed dormancy under both greenhouse and field environments. About 20 crosses were made between the isogenic lines or between the isogenic lines and a male sterility (MS) maintaining line or a MS fertility restoration line. The hybrid F1 plants were obtained and grown in a greenhouse. Recurrent backcrossing with marker-assisted selection will be used to transfer one or more of the s into the male sterility or restoration lines.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Pipatpongpinyo W, Korkmaz U, Wu H, Kena A, Ye H, Feng J, Gu X-Y. 2020. Assembling seed dormancy genes into a system identified their effects on seedbank longevity in weedy rice. Heredity. 124:135-145.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Karakoc Y, Wang J, Gu X-Y. 2019. Mapping of Ehd1-regulated seed dormancy and flowering time genes in rice (Oryza sativa L.). The 12th Annual Plant Science Symposium. University of Minnesota, Saint Paul, MN. March 28-29.
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2019
Citation:
Karakoc Y, Wang J, Gu X-Y. 2019. Mapping of quantitative trait loci associated with seed dormancy and flowering time in rice (Oryza sativa L.). The 35th Annual Plant Science Graduate Student Symposium. North Dakota State University, Fargo, ND. March 15-16.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Gu X-Y, Feng JA. 2019. Transgenerational pattern of epigenetic inheritance for seed dormancy associated with qSD12 in rice. The 6th Workshop on Molecular Aspects of Seed Dormancy and Germination. Volendam, The Netherlands. September 22-25. (Abstract #31).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Karakoc Y, Wang J, Pipatpongpinyo W, Gu X-Y. 2019. Reverse genetic analysis of the Ehd1 gene for the association between seed dormancy and flowering time in rice (Oryza sativa L.). The 2019 ASA-CSSA-SSSA International Annual Meeting. San Antonio, TX. Nov. 10-13.
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