Source: SOUTH DAKOTA STATE UNIVERSITY submitted to NRP
MITIGATING TRANSGENE FLOW USING A LINKED CRISPR/CAS9-BASED MULTIPLEX AGAINST SEED-RELATED ADAPTIVE TRAITS IN WEEDY RICE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1027181
Grant No.
2021-33522-35486
Cumulative Award Amt.
$499,600.00
Proposal No.
2021-04286
Multistate No.
(N/A)
Project Start Date
Sep 1, 2021
Project End Date
Aug 31, 2026
Grant Year
2021
Program Code
[HX]- Biotechnology Risk Assessment
Recipient Organization
SOUTH DAKOTA STATE UNIVERSITY
PO BOX 2275A
BROOKINGS,SD 57007
Performing Department
AHPS
Non Technical Summary
Gene flow from genetically engineered (GE) crops to sexually compatible wild or weedy relatives can exacerbate weed problems when the crops carry a fitness-enhancing transgene. Seeds are deliverables of transgenes. Dormant seeds can survive in the soil over seasons, resulting in weed persistence and the spread of an escaped transgene in agroecosystems. Thus, the goal of this project is to develop a transgenic mitigation strategy by linking a fitness-enhancing transgene with a CRISPR/Cas9-based multiplex system against seed-related adaptive traits to suppress soil seed banks and the spread of the transgene in crop-weed hybrids.The proposed transgenic mitigation strategy will be tested in weedy rice, the most difficult to control weed in rice-growing areas, including the South-Central United States where gene flow from Clearfield® rice has been a concern for more than 10 years. Several genes associated with seed dormancy and its interrelated adaptive traits have been cloned from black-hulled weedy red rice. Functional alleles of the cloned genes have been eliminated from cultivated rice during domestication. Thus, knocking out the weed-specific genes has no effect on crops, but can reduce the fitness of weeds and crop-weed hybrids. Objectives of this project are: 1) to develop transgene-containing CRISPR/Cas9-based multiplex systems for high-efficient mutagenesis of the weed-specific genes in cultivated rice, and 2) to evaluate transgene-mitigating efficacy of the multiplex systems in crop-weed hybrid populations across generations.The transgene Bar for herbicide resistance (HR) and different sets of small guide RNAs (sgRNAs) identified from six weed-specific genes have been (or will be) used to develop transgene-containing CRISPR/Cas9-based multiplex systems. The Bar-containing multiplex systems, like the Bar::Cas9::sgRNAs tandem construct, will be used to transform the rice cultivar Nipponbare. Resulting transgenic lines will be assayed for HR and the copy number of the tandem construct. Single-copy transgenic lines will be evaluated for types and frequencies of site-specific mutations for each of the target genes. The transgenic lines with high-efficient mutagenesis will be crossed with accessions of black-hulled weedy red rice to mimic transgene flow. The hybrid F1s will be evaluated for site-specific mutations and mutant effects on seed traits. The hybrid F2 populations will be used to map the insertion locus of the tandem construct on the genome, to evaluate mutant effects of the multiplex system, and to assess the strengths of the built-in linkage between HR and each of the traits. The F2 seed samples will be used to model the linkage (mitigating) effect on germination dynamics for the target genes expressed in the embryo tissues. F3 families will be selected to model the linkage effect on soil seed bank longevity for the target genes expressed in the seed maternal tissues.Outcomes from the project will include technical parameters and efficacy of a CRISPR/Cas9-based multiplex system on weed adaptation, which can help regulatory agencies and researchers better understand challenges and perspectives of synthetic gene drive in weed control and transgenic mitigation. This project will also produce new genetic materials, such as the transgenic lines and the transgene-free mutants isolated from crop-weed hybrid populations, and will train post graduate researchers. The CRISPR/Cas9-based transgenic mitigation strategy tested in rice could be extended to the other conspecific or congeneric weed-crop systems.
Animal Health Component
20%
Research Effort Categories
Basic
60%
Applied
20%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2131530108050%
2012300114050%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms; 213 - Weeds Affecting Plants;

Subject Of Investigation
1530 - Rice; 2300 - Weeds;

Field Of Science
1080 - Genetics; 1140 - Weed science;
Goals / Objectives
Many crops have conspecific or congeneric weedy relatives in the same agroecosystem. Gene flow from genetically engineered (GE) crops to the weedy relatives may exacerbate weed problems. Seeds are deliverables of biotechnologies, and dormant seeds can survive in the soil for years, resulting in weed persistence and the spread of escaped transgenes from GE crops in weed populations. Synthetic gene driven technologies, such as the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) system, have been demonstrated for the genetic control of disease-vectoring insects. CRISPR/Cas9 has also been proposed for weed management and engineering resilience by editing genes that enhance the persistence of soil seed banks. Thus, the goal of this project is to develop a transgenic mitigation strategy by linking a primary transgene with a CRISPR/Cas9-based multiplex system against seed-related adaptive traits to suppress weed soil seed banks.Weedy rice (Oryza sativa) is an economically important pest in rice-growing areas, including the South-Central United States where gene flow from the herbicide-resistant Clearfield® rice has been a concern for more than 10 years. Most adaptive traits in annual weeds are seed related, such as early shattering, enhanced dormancy, extended soil seed bank longevity, long awns, and dark/red pigments. Our previous research established that seed dormancy associates with each of the other seed-related traits and that the associations arise from pleiotropic or tightly linked genes in weedy rice. Some of the genes have been cloned and characterized for molecular functions, such as SD7-1, SD12a, SD12b and SD12c for seed dormancy, Bh4 for black hull, and Rc for the red or Pp for the purple pericarp color. These genes are functional in weedy but not (or rare) in cultivated rice. Thus, editing of these genes could reduce the adaptivity of weeds, but has little effect on cultivars. The weed-specific genes are selected as editing targets to test the transgenic mitigation strategy in this project. Two objectives of this project are stated below.Objective 1: To develop transgene-containing CRISPR/Cas9-based multiplex systems for high-efficient gene editing of seed-related adaptive traits in weedy rice.Tandem constructs that combine the transgene Bar for herbicide resistance (HR) and a set of small guide RNAs (sgRNAs) from selected targets (SD7-1, SD12a, SD12b, SD12c, Bh4, Rc and Pp), or Bar::Cas9::gRNAs, will be developed to generate built-in linkages between HR and the seed traits. Transgenic lines for the tandem constructs will be produced and purified in the genetic background of a cultivar. Single-copy transgenic (T0 and T1) lines will be analyzed for types and frequencies of CRISPR/Cas9-induced mutations at the target loci. Finally, transgenic T1 lines with HR and the highest editing efficiency for multiple genes will be selected for experiments described in objective 2, as well as for future research. Since the recipient cultivar of the Bar::Cas9::sgRNAs transgenes is a natural mutant for all the target genes, the gene editing efficiency will be evaluated at only the DNA level.Objective 2: To evaluate mitigating efficacy of the multiplex systems in crop-weed hybrid populations.The selected Bar::Cas9::sgRNAs transgenic lines will be crossed with an accession of black-hulled weedy red rice to mimic transgene flow. The hybrid F1 plants will be sequenced for the target genes to identify editing efficiency at the DNA level and also evaluated for the seed-related traits. The hybrid F2 populations will be used to map genomic positions of the transfer-DNAs and to evaluate the built-in linkage strengths of HR with seed dormancy, hull color, and pericarp color. Finally, the F3 progeny lines will be selected and used to model effects of the Bar::Cas9::sgRNAs constructs on germination dynamics and soil seed bank longevity under controlled conditions that mimic the field environments.
Project Methods
Objective 1: To develop transgene-containing CRISPR/Cas9-based multiplex systems for high-efficient gene editing of seed-related adaptive traits in weedy rice.Small guide RNAs: sgRNAs were (or will be) selected from each of the six genes for seed dormancy (SD12a, SD12b and SD12c), for black hull color (Bh4), or pleiotropic for both seed dormancy and red/purple pericarp colors (SD7-1/Rc and SD4/Pb). These genes were cloned from an accession of black-hulled weedy red rice. Different software tools were used to identify sgRNA and PAM (protospacer adjacent motif) sequences from exon or exon-intron junction regions of the genes. Uniqueness of the sgRNA was confirmed by blast search against the reference genome sequence from the japonica-type cultivar Nipponbare.Tandem constructs for built-in linkage: The Bar gene for herbicide resistance (HR) was used as a fitness-enhancing transgene and a Cas9::sgRNAs multiplex for up to 6 target genes was used as a mitigating factor to develop a tandem construct, like the Bar::Cas9::sgRNAs structure. sgRNA expression cassettes were developed using Cas9/sgRNA intermediate vectors driven by the rice small nuclear RNA promoters OsU6a, OsU6b, OsU6c and OsU3.Transformation and transgenic plant analysis: The tandem constructs were (or will be) introduced into the Agrobacterium strain EHA105 to transform the cultivar Nipponbare. Transgenic T0 plants were evaluated for the herbicide resistance and determined for the copy number of the transgene by Southern blotting analysis. The herbicide-resistant single-copy T0 plants (heterozygotes) were advanced to the T1 generation to select homozygotes for independent transgenic events.Estimating multigene-editing efficiencies: The herbicide-resistant single-copy transgenic lines will be used to evaluate types and frequencies of CRISPR/Cas9-induced mutations for each of the target genes. First, the mutations will be evaluated in the Nipponbare background by sequencing the target regions from T1 plants samples from selected transgenic events. The transgenic lines with high mutant rates at the DNA level will be selected to cross with accessions of black-hulled weedy red rice. Then, the hybrid F1 plants will be evaluated for mutant types and frequencies at both the DNA and phenotypic levels. The phenotypes include the degree of seed dormancy associated with all the target genes and the qualitative traits black hull color and red or purple pericarp colors that are controlled by single dominant genes Bh4, Rc or Pb.Objective 2: To evaluate mitigating efficacy of the multiplex systems in crop-weed hybrid populations.Mapping the transgenes: F1 plants with mutant alleles at all the target genes will be advanced to the F2 populations to map a transfer-DNA (T-DNA) insertion on the genome. Thermal asymmetric interlaced PCR or bulked segregation analysis (BSA) will be used to map the T-DNA using the F2 populations. For BSA, an F2 population will be assessed for herbicide resistance (HR) or susceptibility (HS) associated with the Bar gene, and genotyped with codominant markers such as simple sequence repeats (SSR). SSR markers closest to the insertion locus will be used to precisely mark the Bar::Cas9::sgRNAs construct in the following experiments.Estimating gene editing effects of the Cas9-based multiplexes on the seed-related adaptive traits: The segregation of a Bar::Cas9::sgRNAs construct in an F2 population is expected to associate with herbicide resistance or susceptibility with each of the traits due to the built-in linkage. The linkage effects can be best estimated using the marker closest to the T-DNA insertion, as it can distinguish three (hemo-, hemi- and nulli-zygous) genotypes for the transgene. Thus, an F2 population will be genotyped with mapped markers flanking the T-DNA insertion locus and evaluated for HR/HS, the degree of seed dormancy, and hull and pericarp colors. The genotyping and phenotyping data will be used to estimate three groups of genetic effects: 1) for built-in linkage between transgenic (HR/HS) and each of the seed traits, 2) for each of the target genes, and 3) for trait associations.We noticed that the marker genotypes can differentiate between the parental origins of the alleles in an F2 plant, but cannot distinguish the difference between a functional and a non-functional mutation in the F1 or F2 generations. Thus, the F2 plants will be selected for sequencing of the target regions to improve the data annotation and to choose F3 lines for the following experiments.Modeling germination dynamics and soil seed bank longevity: We hypothesize that editing multiple genes for the seed-related traits will negatively impact the persistence of soil seed banks, and consequently mitigate the spread of an escaped transgene. To test the hypothesis the target genes will be divided into two groups based on their expression in the embryo (SD12a, SD12b & SD12c) or maternal (Bh4, Rc & Pb) tissues to model their mitigating effects. The embryo-expressed genes will be evaluated for mitigating effects on germination dynamics of F2 seeds over a period of burial in the soil. A population of newly harvested F2 seeds (5,000) will be sown in moist soil under controlled conditions. The seedlings will be collected every 30 days to evaluate HR/HS, and will then be genotyped for SD12s. The data will be used to model distribution patterns for genotypic frequencies of SD12s for each of the HR and HS groups, and for each of the homo-, hemi- and nulli-zygous genotypes. The maternal tissue-expressed genes will be evaluated for mitigating effects on soil seed bank longevity using F3 families. The F3 families will be selected from F2 plants that are homozygous for the T-DNA insertion, Bh4, and Rc or Pb loci. Duplicated samples of F3 seeds from different families will be buried in the soil for seven months to mimic the field conditions. The buried samples will be evaluated for seed decay rate and survivability. Data from the burial trial will be used to model main and interactional effects of the maternal tissue-expressed genes and the mitigating effects of the Bar::Cas9::sgRNAs construct on soil seed bank longevity.

Progress 09/01/23 to 08/31/24

Outputs
Target Audience:Three groups of target audience were reached in year 3. The first group was seed biologists and plant ecologists in the 7th International Plant Dormancy Symposium at the University of Western Australia from September 11 to 15, 2023. Some of the data from this project were presented in a talk entitled "Three Types of Seed Dormancy and Their Evolutionary and Regulatory Mechanisms in Rice". The second group of audience included scientists and government coordinators in the USDA NIFA Biotechnology Risk Assessment Grants (BRAG) Program 2024 Project Director's Meeting on May 21, 2024. The most recent data from this project was presented in the talk entitled "Mitigating transgene flow using a linked CRISPR/Cas9-based multiplex against seed-related adaptive traits in weedy rice". The third group was faculty members and graduate students who attended two lectures, one at Tasmanian Institute of Agriculture, University of Tasmania on Sept. 18, 2023, and the other at South China Agricultural University on October 15, 3023. The PD was invited to give lectures to review recent progress and applications of research on seed dormancy and weed adaptation. And the other group was the graduate students in the PD's classes "Genome mapping and QTL analysis (PS792)" and "Molecular Plant Physiology (BIOL/PS664". Phenotyping, genotyping and molecular biology data from this project were selected for real-data practices or as examples to explain evolutionary mechanisms of plant adaptation in the classes. Changes/Problems:The F3 population for the 2T system and the F5 population for the 1T system in the east greenhouse were lost in December of 2023 to January of 2024 due to the outage of the heating system. It takes time to recover the populations. What opportunities for training and professional development has the project provided?The doctoral graduates (Kamal Bhattarai, Marya Bibi, Huayu Xu and Bhupinder S Batth) and the postdoctoral research associate Rupak Chakraborty received training in the third year of the project. Marya and Rupak kept working on sequencing and decoding of the mutants. Marya and Kamal worked on developing higher generations of the segregating populations for the 1T and 2T systems. Huayu and Marya worked on bioinformatics to annotate mutant DNA sequences and deduced protein sequences. Bhupinder participated in analysis of the phenotypic and genotyping data. All of them were trained for bioinformatic tools used to decoding and annotating the sequences. How have the results been disseminated to communities of interest?Experimental data from this project were selected to present in: 1) the 7th International Plant Dormancy Symposium at the University of Western Australia from September 11 to 15, 2023; 2) an invited lecture at Tasmanian Institute of Agriculture, University of Tasmania on Sept. 18, 2023, and the other at South China Agricultural University on October 15, 3023; 3) an invited lecture at the South China Agricultural University on October 15, 2023; 4) the USDA NIFA Biotechnology Risk Assessment Grants (BRAG) Program 2024 Project Director's Meeting on May 21, 2024; 5) the class "Genome mapping and QTL analysis (PS792)" in the fall semester of 2023; 6) the class "Molecular Plant Physiology (BIOL/PS664)" in the spring semester of 2024; and 7) a dissertation for a doctoral graduate at SDSU. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: To develop transgene-containing CRISPR/Cas9-based multiplex systems for high-efficient gene editing of seed-related adaptive traits in weedy rice. Identifying mutations induced by the Crispr/Cas9-based multiplex in the hybrid F1 plants to compare the editing efficiency in the background of weedy rice between the two multiplex systems. Objective 2: To evaluate mitigating efficacy of the multiplex systems in crop-weed hybrid populations. Continue to sequence F4 plants from the 1T multiplex system and F2 plants from the 2T multiplex system and annotate the sequences to identify functional point mutations and to compare mutant types and frequencies of the multiplex systems among the six genes in the genetic background of weedy rice. Select mutant plants for progeny testing to evaluate mitigating effects of the 1T and 2T multiplex systems on the weed adaptive traits. Purify mutant lines for each of the six genes for further research in the PD's and other laboratories. Continue to identify mutations induced by the multiplex systems in the two F2 populations plants, to complete genotyping of the two F2 populations and develop linkage maps based on the F2 data for gene-based and SNP mappers, and to complete phenotypic assessment for the seed-related adaptive traits. Keep advancing the F2 populations to higher generations to purify the mutant genotypes and to evaluate the mitigating efficiency more precisely.

Impacts
What was accomplished under these goals? Objective 1: To develop transgene-containing CRISPR/Cas9-based multiplex systems for high-efficient gene editing of seed-related adaptive traits in weedy rice. 100% Accomplished Completed sequence analysis for mutants induced by two (1T & 2T) CRISPR/Cas9-based multiplex systems for five (Bh4, SD7-1, SD7-2, SD12a & SD12c l) of the six target genes in the genetic background of Nipponbare (NB). The 1T and 2T systems contain one and two guiding RNAs (gRNA) for each of the five genes, respectively. A total of 32 T0 plants (64 gametes) from the 1T system and 33 T0 plants (66 gametes) from the 2T system were sequenced for the target regions within the five genes. Three groups of point mutations were observed on the 130 gametes (i.e., deletion of 1 to 20 bp, base insertion & base substitution). The deletion, insertion and substitution mutants accounted for 62%, 33% and 5%, respectively, in the sequenced plants. These plants can be divided into four groups, based on mutations that occurred to one or two of the alleles at a gene/locus in the diploid species: homozygous, heterozygous, biallelic and unknown. The homozygous group of plants contains two identical mutant alleles at a locus, and the frequency varied from 14.8% for SD7-1 to 28.6% for SD12c. The heterozygous group of plants contains one wild-type (WT) and one mutant allele at a locus, and the frequency varied from 0 for SD12c to 42.1% for Bh4. The biallelic group of plants contains two different mutant alleles at a locus, and the frequency varied from 31.6% for Bh4 to 60.7% for SD7-2. The unknown group of plants contains two unknown mutant alleles at a locus, and the frequency varied from 7.1% for SD7-2 to 25.0% for SD12c. The mutant rate was higher for the 2T (88.0%) than for the 1T (79.4) system, on average. Of the 65 sequenced plants, about 13% had no mutation in the 1T and 2T systems, and 56% and 79% had simultaneous mutants at the five loci in the 1T and 2T systems, respectively. The recipient parent NB of the transgenes is a cultivar that contains natural mutant for all the six genes. Thus, it is impossible to determine if the mutations detected in the NB background could alter the genes' functions. In addition, the SD12b alleles from the selected plants were not sequenced because the recipient contains a deletion of 84 bp that was used to design a gRNA for the mutagenesis. Objective 2: To evaluate mitigating efficacy of the multiplex systems in crop-weed hybrid populations. 50% Accomplished The F2 population for the 1T system was advanced to the F4 generation by a single-seed-descent technique. A range of segregations was observed in each of the F2, F3 and F4 populations for: 1) the transgene evaluated by the resistance/susceptibility to the hygromycin or herbicide; 2) the weed adaptive traits seed shattering, awn length and black and pericarp colors; and 3) the agronomic traits plant height and flowering time. A strong segregation distortion was observed for the black hull gene Bh4 from the F2 to F4 populations, with the proportion of the plants having a black hull color (dominance character) much less than the Mendelian expectations. The deviation pattern suggests that some of the Bh4 alleles from the weedy rice parent were mutated by the 1T multicomplex. The F4 population of 192 plants was genotyped with a selected panel of single nucleotide polymorphism (SNP) markers. A linkage map was constructed using the SNP markers. The transgene was located on chromosome 8 using the linkage map. Quantitative trait locus (QTL) analysis for the other traits is on-going. A sample of 10 F4 plants were sequenced for the six target genes, and point mutations were detected for the six genes, including the Bh4 allele from the weedy rice. We are sequencing additional F4 plants, selected based on the SNP genotypes, to identify the 1T multiplex-induced mutations in the advanced generation. The F2 population for the 12 system was advanced to the F3 generation by a single-seed-descent technique. Segregation patterns for the transgene, weed traits and agronomic traits in the F2 and F3 populations were similar those observed for the 1T system. Analyses of the 2T system are on-going, including sequencing, decoding of mutants and QTL analysis.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Xing-You Gu, Marya Bibi, Kamal Bhattarai, Rupak Chakraborty, Hang Yu, Alex Kena, Heng Ye. Mitigating transgene flow using a linked CRISPR/Cas9-based multiplex against seed-related adaptive traits in weedy rice. USDA NIFA Biotechnology Risk Assessment Grants (BRAG) Program 2024 Project Directors Meeting. May 21, 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Xing-You Gu, Kamal Bhattarai, Bhupinder S, Batth, Brent Turnipseed. 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. Perth, Australia.


Progress 09/01/22 to 08/31/23

Outputs
Target Audience:Three groups of target audience were reached in year 2. The first group was seed and plant biologists in the seed biology multi-state project (W4168) meeting in Tucson, AR in October 2022. In this meeting, we reported the seed dormancy genes selected as targets to mitigate gene flow, and the development of transgenic lines for CRISPR/Cas9-based multiplex and hybrids between the transgenic lines and a weedy rice accession. The second group was weed biologists, agronomists, graduate students, and rice farmers, and industry representatives in the 39th Rice Technical Working Group Meetings in Hot Springs, AR from Feb 20-24, 2023. We discussed ecological genetic mechanisms underlying gene flow from ClearField cultivars to weedy rice and the mitigating strategy using the CRISPR/Cas9-based multiplex against seed-related adaptive traits in a presentation to the 2023 conference. The third group was graduate students in the "Crop Physiology" class in the Fall semester 2022 and the Molecular Plant Physiology class in the spring semester of 2023. Changes/Problems:The project was delayed because the two segregating populations of plants in a greenhouse were killed by an incidence of power outage in late December of 2022. The populations were regenerated to keep the project moving ahead as proposed, but the time was delayed. What opportunities for training and professional development has the project provided?Three doctoral graduates (Kamal Bhattarai, Marya Bibi, and Huayu Xu) and a postdoctoral research associate Rupak Chakraborty received training in the project. Kamal and Rupak continued to work on this project part time, and both participated in maker genotyping, collecting phenotypic data, and sequencing of the target genes to identify CRISPR/Cas9-induced mutants. Marya was recruited specifically to work on this project from the fall semester of 2022 and trained for plant cultivation, hybridization, managing plant segregating populations, designing CRISPR/Cas9-multiplex and bioinformatics. Huayu joined this project from the spring semester of 2023 and was trained to conduct seed biological, genetic, and molecular biological experiments. Rupak also assisted with the PD to coordinate the lab and greenhouse experiments, and to train graduate students. How have the results been disseminated to communities of interest?Part of the experimental data were presented in the seed biology Multistate project meeting or the Rice Technical Working Group meeting or will be present in the International Plant Dormancy Symposium in September 2023. Some of the data were selected as real examples to explain the principles and applications of genome-editing technologies in the PD's classes. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: To develop transgene-containing CRISPR/Cas9-based multiplex systems for high-efficient gene editing of seed-related adaptive traits in weedy rice. Identifying mutations induced by the Crispr/Cas9-based multiplex in the hybrid F1 plants to compare the editing efficiency in the background of weedy rice between the two multiplex systems. Objective 2: To evaluate mitigating efficacy of the multiplex systems in crop-weed hybrid populations. Continue to identify mutations induced by the multiplex systems in the two F2 populations plants, to complete genotyping of the two F2 populations and develop linkage maps based on the F2 data for gene-based and SNP mappers, and to complete phenotypic assessment for the seed-related adaptive traits. Keep advancing the F2 populations to higher generations to purify the mutant genotypes and to evaluate the mitigating efficiency more precisely.

Impacts
What was accomplished under these goals? Objective 1: To develop transgene-containing CRISPR/Cas9-based multiplex systems for high-efficient gene editing of seed-related adaptive traits in weedy rice. 90% Accomplished Two sets of hybrid F1 plants were developed from crosses between the Nipponbare transgenic (1T and 2T) lines and an accession of weedy rice, which contain functional alleles for the six target genes. Sequencing of the F1 plants is on-going to identify mutant types and frequencies in the genetic background of weedy rice. The mutagenic data will be used to compare the difference in gene-editing efficiency between the two (1T and 2T) CRISPR/Cas9-based multiplex systems in the hybrid F1 generation. Objective 2: To evaluate mitigating efficacy of the multiplex systems in crop-weed hybrid populations. 30% Accomplished Two sets of crop-weed crosses were made as the above-stated. The resulting hybrid F1 plants were confirmed with marker genotypes and plant morphologies. Mutant phenotypes in the F1 plants were observed for the straw hull color and reduced seed dormancy for the black hull gene Bh4 and some of the dormancy genes. The F2 seeds were used to develop the F2 plant populations segregating for the CRISPR/Cas9-induced mutations and weed adaptive traits. About 350 F2 plants were developed from the two populations. DNA samples from individual plants were collected and genotyped with selected markers located within the target genes. A subpopulation of 94 plants from each of the two populations were also genotyped using a high-throughput SNP array. The marker-genotyping data are being used to: 1) map the transfer-DNA for the Crispr/Cas-9-based multiplex to track the transgene gene across generations; and 2) to identify new mutations in the F2 generation, segregating patterns for the mutations induced in the F1 and F2 generations, and mitigating effects on the seed-related adaptive traits associated with individual and combination of the mutations.

Publications

  • Type: Journal Articles Status: Submitted Year Published: 2023 Citation: Wenwu Tan, Min Guo, Yue Zhu, Rupak Chakraborty, Guiquan Zhang, De-Yu Xie, Xing-You Gu. The Rc and Pb genes regulate seed coat-pigmentation patterns, flavonoids, dormancy and germination in rice. Plant Physiology.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Gu, Xing-You, Kamal Bhattarai, Bhupinder Batth, Brent Turnipseed. 2023. Three types of seed dormancies and evolutionary and regulatory mechanisms in rice. The 7th International Plant Dormancy Symposium, The University of Western Australia, Sept. 11-15.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Xing-You Gu, Ahmed Charif, Bhupinder Batth, Brent Turnipseed. Modeling G-by-E interactions for seedbank. 2023. Longevity of weedy rice in till and no-till cropping systems. The 39th Rice Technical Working Group Meeting, Hot Springs, Arkansas Feb. 20-24.
  • Type: Journal Articles Status: Published 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. Eds. Upadhyaya, M.K., Clements D.R., and Shrestha A. Wiley. NY, NY.


Progress 09/01/21 to 08/31/22

Outputs
Target Audience:There were two groups of the target audience for year 1. The first group included attendees at the annual meeting of multi-state project for seed biology held at Cornell AgriTech, Geneva, NY in mid-October and the XXIX Plant and Animal Genome (PAG) Conference held online on 7-12 January 2022. Professors, researchers, and graduate students working in the areas of seed biology/technology or agronomy from universities or state agricultural extension centers attended the multi-state annual meeting. During the meeting, we introduced the objectives, preliminary data, and experimental plans for the new project by an oral presentation and personal communications. Attendees to the PAG conference included scientists and researchers working in areas of genetics, genomics, molecular biology, and agriculture. We introduced some preliminary data for the new project to the conference by two abstracts and two e-posters. The second 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 12 graduate students in the spring semester of 2022. The PD selected some of molecular biological, genomic, or quantitative genetic data as examples for real data practices or for lectures to explain applications of CRISPR/Cas9-based genome editing techniques in agriculture or plant biology. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A postdoctoral research associate (Dr. Rupak Chakraborty) and two Ph.D graduate students (Bhupinder Batth and Kamal Bhattarai) worked on the project in the first year. The postdoc was trained for plant molecular biology, including vector construction, sequencing, and sequence analysis. The two graduates were both trained for bioinformatics, DNA samples preparation for sequencing, transgenic plant analysis using the hygromycin assessment, maintaining the transgenic plants and seed samples, and hybridization. How have the results been disseminated to communities of interest?The objectives, rationale, and some preliminary data were presented in the Seed Biology Annual Meeting or the Plant and Animal Genome conference. The strategy to mitigate transgene flew using a CRISPR/Cas9-based multiplex system against seed adaptive traits was discussed in a chapter of the new book for weed scientists and in the PD's classes for graduate students. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: To develop transgene-containing CRISPR/Cas9-based multiplex systems for high-efficient gene editing of seed-related adaptive traits in weedy rice. Purify the transgenic T1 and T2 lines by self-pollination, single plant selection, and hygromycin assessment. Some of the pure lines may be selected for sequencing to identify types and frequencies of mutations. Objective 2: To evaluate mitigating efficacy of the multiplex systems in crop-weed hybrid populations. Develop hybrids between the transgenic T1 or T2 lines and lines of weedy rice. Advance the F1 hybrids to the F2 and higher generations, track the segregation patterns of the CRISPR/Cas9-based multiplex across the generations, map the transgene insertion site, evaluate seed-related adaptive traits in the F2 and F3 populations, and identify the types and frequencies of mutants at the six target genes from the parent of weedy rice in the hybrid populations.

Impacts
What was accomplished under these goals? Objective 1: To develop transgene-containing CRISPR/Cas9-based multiplex systems for high-efficient gene editing of seed-related adaptive traits in weedy rice. 70% Accomplished Two tandem constructs were developed to edit six (6) genes for seed-related adaptive traits, i.e., Bh4, SD7-1, SD7-2, SD12a, SD12b and SD12c. The two multiplex constructs contain the same genes for the herbicide resistance (Bar) or CRISPR-associated protein 9 (Cas9), but differ in the number of small-guide RNAs (sgRNAs). One tandem construct contains one sgRNA for each of the 6 genes, like Bar::Cas9::6sgRNAs or TC6x1. The other multiplex has two sgRNAs for each of the 6 genes, Bar::Cas9::6x2sgRNAs (TC6x2). The two sgRNAs for each gene were designed to target different sites of the coding sequence to increase the CRISPR/Cas9-induced mutant frequencies. All the coding sequences were cloned from weedy rice. TC6x1 and TC6x2 were confirmed by sequencing before transformation. The two constructs were used to transform the japonica-type cultivar Nipponbare, a natural mutant for all the six genes. A total of 97 transgenic T0 plants were obtained, which represent five independent transgenic events for each of the two constructs. All the plants were determined for the presence of the transgenes by the hygromycin assay at the seedling stages and recorded for agronomic traits and seed setting percentages. Southern blotting analysis was used to confirm the transgenes. Mutant analysis was conducted for the T0 plants sampled from different transgenic events. For each of the two multiplex constructs, about 30 T0 plants were selected to sequence target sites in each of the 6 genes. The sequences were decoded to identify types and frequencies of mutants. Of the 6 genes, SD12b was failed to decode sequences for both the constructs. The Bh4, SD7-1, SD7-2, SD12a and SD12c genes showed three groups of point mutations: deletions (1-20 bp), insertion (1-2 bp) and substitution (A/T or G/C), which accounted for 62%, 33% and 5% of the total mutations, respectively, at the sequence (haploid) level. The frequency for the mutations simultaneously occurred at all five genes were higher for TC6x2 (78%) than for TC6x1 (56%). The frequency for no mutation to occur at the 5 genes was 10% for the two constructs. Of the five genes, Bh4 had a higher mutant rate in TC6x2 (88%) than in TC6x1 (60%) and the remaining were similar in the mutant frequency (80-90%) between the two constructs. There were four types of mutants the plant (diploid) level: biallelic (46%), heterozygous (22%), homozygous (21%), and unknown (11%). About 60 T1 lines were selected to purify the mutants in the genetic background of Nipponbare. Objective 2: To evaluate mitigating efficacy of the multiplex systems in crop-weed hybrid populations. 5% Accomplished Hybridization was made between a few T0 plants and an accession of weedy rice (SS18-2). The hybrid F1 plants are being grown for mutant analysis and to develop the F2 populations for subsequent research.

Publications

  • Type: Journal Articles Status: Awaiting Publication 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. Eds. Upadhyaya, M.K., Clements D.R., and Shrestha A. Wiley. NY, NY.
  • 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, B.S., Turnipseed, E.B., 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.