Progress 02/15/19 to 02/14/23
Outputs
Target Audience:Through the reporting period, I provided formal research experiences to four Chapman University Undergraduate students and informal summer research internships to six high school students. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided valuable research training opportunites to Chapman University undergraduate students as well as high school students through summer research internships. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Acknowledgment With this being the final report, I would like to thank the USDA-NIFA for funding this project. This grant gave us the opportunity to generate valuable results in terms of breeding new chia (S. hispanica) varieties adapted to US environmental conditions. I believe we were able to generate the neededgenomic and phenotypic resources as part of this SEED grant to apply for a full grant this October.Thisgrant also provided research training to several undergraduate and high school students.I want to thank the former National Program Leader, Dr.EdwardKaleikau,for the helpful discussions and for his willingness to provide two years of no-cost extension for me to recover from the pandemic-related setbacks. Finally, I look forward to interactingwith the new National Program Leaders, Dr. Christian Tobias and Dr. Ann Stapleton. The accomplishments during the 2022-2023 reporting period: A) We phenotyped the F4population during the summer of 2022and collected additional phenotypic data.Last year, we noticed that oneF3 line (labeled 6-24) in our breeding population showedsigns of drought tolerance.During the summer of 2022, we conducted a small pilot drought assay in the fieldand confirmed the drought tolerance phenotype of this line. We obtained similar results in pilot greenhouse experiments. We also showed a wild population of chia that was collected from Northern Mexico(labeled N3) was more drought tolerant than the commercial variety pinta.Finally, we measured the transpiration rate, photosynthesis rate, andstomatal conductance. Our results showed that the drought tolerance mechanism in the 6-24 line might be different than the drought tolerance response we observed in the N3 wild accession. We are very excited about these preliminary results, and we will repeat this greenhouse experimenton a larger scale this summer. If successful, we will use this data as one of the preliminary results in a newproposal that we are preparing to submit to thePlant Breeding for Agricultural Production program (code:A1141) dueOctober 13, 2023. B1) Related to the drought assay results (section A), we went ahead and made crosses between the commercial variety Pintaand the N3 wild accession, and bulked the F2 seeds. This F2 population will be used to map the genes responsible for the drought tolerance response observed in the N3 accession. B2) We also crossed the 6-24 line with the N3 wild population.We are currently bulking F2 seeds fromthis cross. Since our preliminary results showed that 6-24 and N3 might have different drought tolerance mechanisms, we hypothesize that combining these different mechanisms in a single plant willproduce an even better drought-tolerancephenotype. C1) Over two seasons, we conclusively demonstrated that the N3 genotype (described in section A) flowers around 40 days earlier than the commercial Pinta variety in Southern California.During the summer of 2022, we used the F2 breeding population described in SectionB1 to conductthe first field trial with 3000 F2 plants and saw the expected phenotypic segregation in flowering time. We collected seeds and leaf samples from the 20 earliest and 20 latest flowering plants. We already have the genome sequence of Pinta. We went ahead and sequenced the genome of the N3 genotype as well and identified variations between the two parents.We will use this population to incorporate the early flowering phenotype in the commercial pinta genotype and find molecular markers linked to the early flowering phenotype of N3. C2) In summary, the wild accession N3 has two phenotypes of interest, namely early flowering and drought tolerance. However, this N3 genotype (like every wild chia accession) hasan open calyx phenotype that limits breeding with wildgenotypes. Open calyx, while beneficial in evolutionary terms, is not desirable for agricultural production purposes as the seeds will disperse. Therefore, we also used our F2 population from this N3 x pinta cross to collect seeds and leaf samples from plants that had closed calyx. We know many genes control this trait, and we expected to observe a quantitative phenotype. Interestingly, out of the 3000 F2 plantsphenotyped, we found four plants with closed calyx similar to the commercial pinta parent. Currently, we are growing seeds from these four plants in the greenhouse to confirm their closed calyx phenotype. Once confirmed, these plants will be used to generate markers for closed calyx phenotype, which will be very helpful for future breeding efforts using wild chia accessions with other desirable characteristics. D) Although the United stateis the biggest importer of chia seeds in the world, chia is not commercially produced in the US. We used 20-year historical climate data to identify regions in the continental US with a suitable climate for commercial chia cultivation. Our analysis in the publication (Hassani et al., 2022) showed that the commercial chia variety can be planted on approximately 1,000,000 hectares of cropland. The future development of early flowering variety (SectionC1) was demonstrated to open an additional 4,400,000 hectaresfor chia cultivation in the United States. E) In section B of last year's report, I mentioned that we extracted and sequenced DNA from 10 plants showing early flowering phenotype and 10 plants showing late flowering phenotype. In addition to 10 plants with big seeds and 10 plants with small seeds. I would like to remind the reader that this F2 population was generated during the first year of the project by crossing pinta with another domesticated variety called tropical. The tropical genotype flowers very late (January in southern California) but has bigger seeds, while the commercial variety pinta flowers relatively early (September in southern California) but has smaller seeds. Our bulked segregant analysis this year with the flowering time trait predicted a mapping interval of size 11.50 Mbp on chromosome 1 harboring the genes responsible for the early flowering phenotype in Pinta. These were based on the summer 2021 samples. We are currently analyzing the samples that we collected in the summer of 2022 and hoping to both confirm the prediction and shorten the mapped interval. Summary of what I promised to deliver in 2021 and what I was able to deliver in 2022 As part of the 2021 annual report, I promised to deliver the following: 1) Analyze the sequences for the Bulk Segregant Analysis and publish the results. The analysis was done (please see section E), and we are trying to refine the mapping interval. The manuscript is expected to be submitted for publication in October. 2) Continue our screening with the F4 generation plants over the summer of 2022 and collect phenotypic data. We made extensive progress in this aim and generated two more F2 breeding populations to study even earlier flowering time, drought tolerance, and closed calyx (sections A, B, and C) 3) Generate sequencing data foradditional traits (trichome density and inflorescence length) and perform Bulk segregant analysis. We collected the samples but did not do the sequencing. 4) Publish the chloroplast genome and the comparative analysis results. We made significant progress on this objective but haven't submitted the manuscript yet. The manuscript is 80% written and all the analysis are completed. We anticipate submission by the end of June. 5) Publish the Salvia hispanica genome sequences generated during the first year. The pandemic set us behind on this, and a Chinese group published the first genome sequence of chia before us. The plan is to publish our genome sequencing and assembly results together with the mapping data that we generated for the early flowering phenotype. 6) Publish our results as the first report of Macrophomina phaseolina infecting chia plants in U.S. We are still working on this objective, and wehaven't submitted the manuscript yet. We are doing a final experiment, and we anticipate submission byAugust.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Hassani, M., Piechota, T., & Atamian, H. S. (2022). Prediction of Cultivation Areas for the Commercial and an Early Flowering Wild Accession of Salvia hispanica L. in the United States. Agronomy, 12(7), 1651.
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Progress 02/15/21 to 02/14/22
Outputs
Target Audience:Through the reporting period, I provided formal research experiences to Chapman Undergraduate students and informal summer research internships to high school students. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided valuable research training opportunites to Chapman University undergraduate students as well as high school students through summer research internships. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period we will do the following and include the results in the Final report in February 2023: 1) Analysethe sequences for the Bulk Segregant Analysis and publish the results. 2) Continue our screening with theF4 generation plants over the summer 2022 and collect phenotypic data. 3) Generate sequencing data for additonal two traits (trichome density and inflorescence length) and perform Bulk segregant analysis. 3) Publish the chloroplast genome and the comparative analysis results. 4) Publish the Salvia hispanica genome sequences generated during the first year. 5) Publish our results as first report of Macrophomina phaseolina infecting chia plants in U.S.
Impacts
What was accomplished under these goals?
A) We phenotyped F3 population during the summer 2021 and collected additional phenotypic data for the following characters segregating in the population. 1) Flowering time 2) Seed size 3) Lodging 4) Calyx trichome density 5) Disease resistance 6) Drought tolerance 7) Inflorescence length B) We also extracted DNA from total of 40 plants for Bulk Segregant Analysis (see below). The samples from each phenotypic category were combined together and a total of 4 samples were sequences using the Illumina sequencing platform. The goal is to generate genetic markers linked to these two triats (flowering time and seed mass). 1) 10 plants showing early flowering phenotype 2) 10 plants showing late flowering phenotype 3) 10 plants with big seeds 4) 10 plants with small seeds. C) We assembled the chloroplast genome of seven Salvia hispanica accesions (4 domesticated and 2 wild) and performed comparative analysis with seven salvia species for which chloroplast genome sequences were publicly available. In addition, we tabulated codon usage bias in salvia species and identified optimal codons that will be useful for future genome engineering projects. D) We identified Macrophomina phaseolina causing the charcoal rot disease in chia. We characterized the pathogen morphologically and molecularly through sequencing.
Publications
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Progress 02/15/20 to 02/14/21
Outputs
Target Audience:The target audiences that I reached during this reporting period were Chapman Undergraduate students and High school student from Orange County. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Throughout the project, I provided training opportunities to threeChapman Undergraduates and two high school students. Kylie Deer (High school student) participated in the phenotyping and DNA extraction of 110 plants. Kevin Nguyen, David Yang (Chapman Undergraduates) and Aditi Jain, Andrea Wang (High school students) participated in the codon usage analysis of the chloroplast genomes over the summer. Contact information Kylie Deer:deer@chapman.edu Kevin Nguyen:kevinnguyen@chapman.edu David Yang:dayang@chapman.edu Aditi Jain:aditij714@gmail.com Andrea Wang:andreawang04@gmail.com How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period we will accomplish the following: a) Publish the nuclear genome of Salvia hispanica to provide the scientific community with the first complete chromosomal scale genome sequence information for any salvia species to date. b) Publish the codon usage bias analysis which will be useful for future genetic engineering projects. c) Perform bulk segregant analysis with the extracted DNA to develop markers forfor seed mass, inflorescence length, flowering time, and trichome density on the calyx. d) Screen the new F2 population (described above)for early flowering and closed calyx and develop genetic markers. e) Perform one last field trial with the F4 population from the'Chia pinta' and'Tropic' cross described above. This will enable us to stabilize the different traits in the selected plants from F3 population which will be used for multi-location field trials in future projects depending on funding.
Impacts
What was accomplished under these goals?
Under the objective 1: a) We continued performing analysis of the genome sequences that we generated during the first year and getting closer to publishing our results. b) Independent of nuclear genome analysis mentioned above, we performed comparative chloroplast genome analysis among the Salvia species and its close relatives and identified the codon usage bias in Salvia hispanica. Under objective 2: a) We collected 2nd year of phenotypic data from the F3 population derived from a cross between 'Chia pinta' (the most commonly used domesticated variety) and a domesticated cultivar having character traits representative of tropically adapted cultivars 'Chia Tropic'. b) We extracted DNA from plants in preparation for bulk segregant analysis with the aim of generatingpreliminary genetic markers for seed mass, inflorescence length, flowering time, and trichome density on the calyx. c) We generated F2 population derived from a cross between 'Chia pinta' and a unique wild plant collected from Northern Mexico that has early flowering characteristic.
Publications
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Progress 02/15/19 to 02/14/20
Outputs
Target Audience:My target audiences during this reporting period included 10 Biology undergraduate students, 20 High-school students, 2 graduate students and 2 postdocs who participated in the different aspects of this project. I provided hands on training to my target audiences through internships and extension and outreach. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Through this project, I was able to provide training and professional development opportunities to a number of adience groups. I provided training to two postdoctoral scholars in my lab. I gave my postdocs some reposnsibilites of running the lab with my supervision. I believe this is a valuable training for them to run their own labs one day as part of their future career goals. They were also involved in mentoring the large number of undergraduate and high schools students that interned in my lab over the past year (details below) I provided training to two graduate students in my lab. My graduate student (James Wimberley) is the first author on the publication that came out of this project so far. My other graduate student (Dony Ang) is currently preparing two additional manuscripts. My lab provided research experience to 10 Chapman undergraduate students during the 2018-2019 academic year. Students participated in this USDA funded project as well as in small independent projects. As a result, 4 students presented posters during the Undergraduate Research Symposium at Chapman University. I intentionally recruit Freshman students so that they are exposed to research environment very early on in their studies. At the Freshman level, my purpose is not to get them involved in big projects at first but rather have them get a feel of research by involving them in a number of ongoing projects in the lab. At the end of the first year (for those who wish to continue in my lab), I design a project with the student tailored towards their interests and feasible in scope. My goal at this stage is to provide them the opportunity to conduct independent research. Most of my students continued the internship for another semester. One of my students, Cailyn Sakurai (Junior), was planning to go to medical school. After working with me for a year, she decided to go for a career in research and at the end of the Spring semester she changed her major to Biochemistry.To me it is very rewarding when I witness students making decisions about their future careers after I provide them research experience. I provided research experience to underserved high school students in my community. The Orange High School is an underserved school with minority enrollment of 93% (majority Hispanic). During the past year, I provided research experience and training to a group of nine Orange High School students (5 males and 4 females). The students worked in my lab one day a week after school between January-June in two groups of 5 and 4 students. Beginning of each meeting, students were given a brief lecture explaining the context of the work they will be doing. During the six months, students got opportunities to plant and maintain chia plants, extract DNA from leaves, perform PCR and run gel electrophoresis. My lab also participated in last year's Simon-Orange-Chapman STEM Scholars (SOCSS) program. As part of this program, 3 students (2 males and 1 female) worked with me over 5 weeks (3 hrs/week) after school. These meetings consisted of 30 min lecture followed by 2 hours of hands on work and final 30 min discussion about the results obtained. Students designed primers, extracted DNA, performed PCR, and ran a gel. Two of the students enjoyed the experience and returned to get more research experience during this summer. In addition, I hosted three students as part of my summer internship program from Woodbridge High School, San Clemente High School, and Diamond Bar High School. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?After we successfully generated all the genomic resources and one round of phenotypic data that we planned for as part of the 1st goal, during the next recording, we will work on connecting the genotype to phenotype. The overarching purpose of this project is generating the phenotypic and the genotypic resources necessary for future Genomics-assisted breeding (GAB) efforts by us and the scientific community. GAB approach results in fast and accurate crop improvement. Before the stay-home orders, my students were extracting DNA from all the plants from which we collected phenotypic data. The plan for the next reporting period is to provide proof of concept results showing with what certainty a given phenotype can be predicted from the genotype of a given plant. In other words, we plan to generate molecular markers associated with the important agronomic characters of chia such as density of trichomes, apical dominance, flowering time, etc. Now that we have a list of all the single nucleotide polymorphisms (SNPs) between the parental varieties, we will systematically find their correlation with the phenotypes collected. During this summer, we will phenotype another 1000 F2 plants from each generation as second replicate. Despite the lockdowns, I fortunately was able to secure a land to conduct my summer experiments according to the planned schedule. While we are generating markers based on the data we have collected during the 1st year, we will collect another round of phenotypic data between June and November. During December to February, we will test our markers on the new set of phenotypic data for confirmation and improvement of our markers. For this summer, I am setting up remote summer internship opportunities to undergraduate and high school students. My plan is to involve them in the computational aspect of this funded project.
Impacts
What was accomplished under these goals?
As part of objective 1: Click on the link below for the figures associated with the results described in this section. https://de.cyverse.org/dl/d/879B5E28-3650-491D-8750-0EB6061A7768/USDA_annual_report.pdf We generated high quality chromosome-scale genome sequence of S. hispanica variety 'Pinta'. a) The total size of the assembled genome was 413 Mb, which is very close to the genome size that has been predicted using flow cytometry. The total number of genes that we annotated with high confidence was 47,335. b) Figure 1 shows the results of the segmental duplications analysis that we performed on the chia genome. We identified around 95,000 segmental duplications between 1000-2000bp long with much less duplications of bigger segments. c) Figures 2 and 3 show the results from our analysis of the syntenic regions between chia and two other closely related genomes sesame and Salvia splendens. The table 1 shows the main statistics from this synteny analysis. Higher percentage of the chia genome (80.17%) had syntenic blocks with Salvia splendens compared to 26.29% with sesame. This is expected as both Salvia splendens and chia (Salvia hispanica) are in the same genus. However, the interesting finding that we will look into in more detail is the fact that most of the chia genome was represented in sesame but not vice versa. This means that a huge genome expansion took place after sesame and chia spatiated from their last common ancestor. d) Table 2 shows the results of orthologous gene analysis. We compared the genes of nine plant species among each other to find the orthologous relationship of their genes. Using this analysis we found genes that have common orthologs among the different species and those that are lineage or species specific. The yellow highlighted cells show that the most orthologous genes were found between Salvia splendens and Sesame, consistent with the syntany results. e) Table 3 shows the analysis of the transposon families in the chia genome. Based on our analysis, 40.86% of the transposons in the chia genome belonged to the Helitron transposon superfamily. Helitrons were reported to be more abundant in gene-poor regions of Arabidopsis. While in maize they mainly exist in the gene-rich region rather than the gene-poor region. We will study the evolution and organization of this transposon family in detail as It is one of the most important agents in gene evolution and can change many gene functions and cause phenotypic differences. We generated shotgun Illumina sequences from S. hispanica variety 'Tropic' and S. hispanica variety 'Wild'. We used the DeepVariant program to identify single nucleotide polymorphisms (SNPs) and insertion/deletions (Indels), by comparing the shotgun Illumina sequences from the 'Tropic' and 'Wild' varieties to the "pinta" genome used as reference. The results are presented in figures 4 and 5. As part of objective 2: We phenotyped 700 plants from segregating F2 population that was derived from a cross between 'Chia pinta' (the most commonly used domesticated variety) and the other domesticated cultivar "Tropic" having character traits. We also phenotyped 120 plants from another segregating F2 population derived from a cross between 'Chia Pinta' and a wild type similar to wild populations from the northern extent of the species wild range 'Chia wild'.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
James Wimberley, Joseph Cahill, and Hagop S. Atamian. De novo Sequencing and Analysis of Salvia hispanica Tissue-Specific Transcriptome and Identification of Genes Involved in Terpenoid Biosynthesis. Plants (9) 405; doi:10.3390/plants9030405
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