Progress 01/15/21 to 01/14/25
Outputs
Target Audience: Plant lipid biochemists and other plant scientists Biotechnology and oilseed businesses Members of the local community interested in agricultural research Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The funding supported the training of one postdoctoral researchwhocharacterizedhigh-linolenic camelina mutants. One graduate student was trained in genomics whose work included genome assemby and identification of candidate genes for fatty acid desaturation.Another graduate student worked on the effects of high temperature during reproductive development on the accumulation of seed fatty acids and other related traits in camelina. One graduate student has successfully defended his thesis and was offered a job in a biotechnology company. One undergraduate student gained research experience in plant science while assisting the graduate student in data collection and analysis. How have the results been disseminated to communities of interest?Results were presented by the graduate students in departmental seminars and three internationalconferences. Two manuscripts have been published in peer reviewed journals with Open Access. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We have achieved these major accomplishments for this project. Objective 1: Isolation of QTLs and candidate genes for fatty acid desaturation and heat tolerance in natural variations 1.1 QTL and candidate genes for fatty acid composition By genome wide association studies (GWAS) using a collection of 212 accessions, we detected a QTL (qOA1.1) for oleic acid (18:1) content on Chromosome 1 that explained over 11% of phenotypic variation (PVE). Attempting to pinpoint the candidate genes was challenging, however, due to a large range of the QTL that encompasses in the chromosome. To improve the resolution and fine map this QTL, two approaches were taken: First, we constructed a chromosome-level reference genome of the camelina variety Suneson using the latest PacBio sequencing and HiC technologies. Recalling genetic markers using the resequenced genomes of the germplasm collection yielded nearly 5.5 million SNPs and over 412,000 InDel markers. Our GWAS with the high-density markers not only detected the same SNP marker associated with 18:1, but also a second QTL (qOA1.2) at a nearby location with an InDel marker. In addition, new markers also enabled for the detection of QTLs that did not appear in the previous studies for other fatty acids such as linoleic acid (18:2) and linolenic acid (18:3). Second, to capture more phenotypic variation under different growing conditions, we grew the population in two environmentally contrasting locations (Sidney MT and Pullman, WA) under two soil nitrogen levels (0 or 100kg/ac nitrogen fertilizer applied) for each location. Interestingly, the SNP marker associated with 18:1 (qOA1.1) was detected at the same chromosomal location in all test environments, suggesting the role of qOA1.1 in determining the 18:1 content in camelina seed. The qOA1.2 was detected in both locations and growing conditions as well, with a PVE of 27% associated with the data from Sidney, MT and a PVE of 25% associated with the Pullman, WA data. 1.2 Phenotypic changes by heat stress To evaluate the impacts of high temperature during seed production, we treated camelina plants in the growth chamber with a temperature set at 22°C (control) or 35°C (heat). Based on the heat effects on multiple traits, we chose 14 days heat treatment, as at this duration the maximal effects on seed yield traits were observed without irreversibly damaging the plants. We found that heat stress significantly reduced fertility during flowering and inhibited storage product biosynthesis and accumulation during seed filling which resulted in smaller and lighter seeds. Oil content decreased while protein content increased in seeds from heat treated plants. In addition, fatty acid composition was altered. Heat stress primarily affected the contents of oleic acid (18:1) and its derived polyunsaturated linoleic acid (18:2) and a-linolenic acid (18:3) in camelina seeds, with the reduction of 18:3 and concomitantly increased 18:2 being the most significantly affected. We also grew the Suneson/Pryzeth derived RIL population of 257 lines in the heat chamber. The distribution of 18:3 contents in the heat-treated seeds showed a normal distribution but a significant shift toward lower contents with a range of 15%-32% than plants grown under control conditions ranging from 25.5% to 39.5%, indicating the negative impacts of high temperatures on 18:3 accumulation. Using the newly developed reference genome, we constructed a high-density genetic map containing 68,791 SNP and InDel markers. This led to the identification of a significant QTL on chromosome 6 (qLNA6.1) for 18:3 besides other QTLs for 16:0 and 20:1. However, in heat-treated plants no QTL was detected for 18:3, but QTLs were detected for 16:0, 18:0 and 20:1. These results suggest that it is possible to identify mechanisms of 18:3 synthesis in camelina seeds and their responses to high temperature by QTL analysis. 1.3 QTL and Transcriptomic analyses of reproductive development under heat treatments Elucidating the genetic response to high temperatures is essential for successful breeding of heat tolerant camelina varieties. We used a combinatorial approach to identifying candidate genes associated with heat stress by QTL mapping and comparative transcriptome profiling. A population of 257 RILs was grown in a controlled growth chamber as described above under the high temperature regimes for 14 days beginning at the onset (with two open flowers) of the reproductive stage. Several traits related to seed production were evaluated at maturity. The QTL analysis identified several regions with co-located traits on chromosomes 8, 10, and 12. Two RILs with contrasting phenotypic responses to heat stress were chosen for gene expression profiling via RNA sequencing. Multiple pathways and genes were found to be strongly affected by heat stress, and many genes expressed differently between the two RILs. Several genes (e.g., bZIP28, HSP21) identified within the QTL regions were considered strong candidates that may control heat tolerance during reproduction in camelina. Objective 2: Discovering novel mechanisms controlling ALA accumulation in seed We used the mutants that contained increased 18:3 to elucidate molecular mechanisms controlling ALA accumulation in seed. We adopted the MutMap strategy by backcrossing the mutant line (e.g., L23335 containing about 43% 18:3) with Suneson (33% 18:3), the wildtype variety used for mutagenesis and for the high-quality genome sequencing. We analyzed the fatty acid composition in the progeny F2 seeds. Although the number of F2 seeds with 18:3 above 39% (38 seeds), 35-39% (88 seeds) and below 35% (35 seeds) fit the Mendelian segregation ratio of 1:2:1, indicating a single dominant mutation that caused the high-18:3 phenotype, the quantitatively continuous variation in the F2 seeds suggests the possibility of EMS-induced mutations at multiple genes. We then performed the bulked segregant analysis (BSA) by whole genome sequencing of DNA from two bulked populations, each comprised of 20 individual plants of high (mutant type) or low (wild type) 18:3 levels. The parental line (L23335) was also sequenced at 20X coverage. The sequences were aligned to the reference Suneson genome to detect SNPs between mutant and wild type using the established bioinformatics tools. To identify mutations responsible for the high 18:3 phenotype or mutations closely linked to the causal mutations, we only retained variants that were consistent with EMS action (CG>TA transitions). We identified two QTLs with the 95-99% confidence intervals: qLNA10 was localized in the genomic interval of 21,697 - 2,848,800 bp with a deltaSNP-index peak of 0.59 at 817,858 bp on chromosome 10, qLNA17 in the interval of 4,903,424 - 5,913,358 with a peak of -0.39 at 5,605,295 bp on chromosome 17. Objective 3: Testing candidate genes Mapping experiments identified two QTLs (qOA1.1 and qOA1.2) on chromosome 1 that may contribute to oleic desaturation. More importantly, these QTLs were repeatedly detected under different environmental conditions at two geographically diverse locations and two nitrogen fertilization regimes. We therefore focused on qOA1.1 and qOA1.2 for further studies. We identified a few candidate genes, including Csa01g013860,a homolog of the Arabidopsis gene At3g12730encoding a putative Myb family transcription factor, PHL12. Two flanking genes including Csa01g013850, an At3g12720 homolog encoding the putative transcription factor MYB67, and Csa01g013870, the Arabidopsis homolog At3g12740 encoding ALIS1, or ALA (aminophospholipid ATPase)-interacting subunit1. We have obtained Arabidopsis T-DNA insertion mutants to test whether there are any effects on seed fatty acid composition. Constructs have been made for overexpression and CRISPR knockout experiments in camelina. We will characterize transgenic plants leveraging other resources since this project has closed.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Smith BE, Lu C (2024) Heat stress during reproductive stages reduces camelina seed productivity and changes seed composition. Heliyon 10 (4):e26678.
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Smith BE, Kemmer S, Decker S, Lu C (2024) Quantitative trait locus (QTL) mapping and transcriptome profiling identify QTLs and candidate genes associated with heat stress response during reproductive development in Camelina sativa. Food and Energy Security 13 (1):e531.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Samuel Decker, Chaofu Lu. Genomic Resources to Understand Genetic Mechanisms of Oil Biosynthesis in Camelina Sativa. International Symposium of Plant Lipids. July 2024, Lincoln, NE
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Samuel Decker, Maral Etesami, Chengci Chen, Chaofu Lu. Identification of QTLs and Candidate Genes for Camelina Nitrogen Use Efficiency and Oilseed Traits Using a High-quality Genome. International Camelina Conference. July 2024, Lincoln , NE
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Brian Smith, Sunny Kemmer, Samuel Decker, Chaofu Lu. Heat stress during reproductive stages reduces camelina seed productivity and changes seed composition. American Society of Plant Biologists, June 2024, Honolulu, HI.
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Progress 01/15/23 to 01/14/24
Outputs
Target Audience:Plant lipid biochemists and other plant scientists Biotechnology and oilseed businesses Members of the local community interested in agricultural research Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The funding has supported the training of one postdoc who is working on the characterization of high-linolenic camelina mutants, and one graduate student who worked on the effects of high temperature during reproductive development on the accumulation of seed fatty acids and other related traits in camelina. Both also received training in genomic sciences such as genetic mapping and transcriptome analyses. The graduate student has successfully defended their thesis and was offered a job in a biotechnology company. One undergraduate student gained research experience in plant science while assisting the graduate student in data collection and analysis. How have the results been disseminated to communities of interest?Results were presented by the graduate studentindepartmental seminar. Two manuscripts havebeen submitted, and another is being preparedfor publication in scientific journals. We areplanning to present some of the results at upcoming conferences in the near future such as Plant Biology 2024 and International Symposium on Plant Lipids. What do you plan to do during the next reporting period to accomplish the goals?The next reporting period is the no-cost extension of this grant. We will therefore focus on Objectives 2 and 3, and the most promising results of last year. We will finish the mapping of QTLs for high-ALA mutant by bulk segregant analysis and finalize the short list of candidate genes. These candidate genes will be tested by gene expression analysis, and if possible, by CRISPR gene knockouts and/or overexpressing studies. While we are generating multiple transgenic plants to test their effects on fatty acid composition in seed, we aim to finish characterizing at least one candidate gene, e.g., MYB67 or the most promising gene based on preliminary results we anticipate obtaining within the first quarter.
Impacts
What was accomplished under these goals?
We have achieved these major accomplishments during this reporting period (1) the identification of QTLs and candidate genes related to fatty acid metabolism, and (2) phenotypic and transcriptomic changes of plants subject to heat treatment during reproductive stages. We have submitted two manuscripts and are preparing for the third for publications. Objective 1: Isolation of QTLs and candidate genes for fatty acid desaturation and heat tolerance in natural variations 1.1 QTL and candidate genes for fatty acid composition Previously, we conducted a QTL mapping using a collection of the diverse germplasm of 212C. sativalines and an advanced population of 257 recombinant inbred lines (RILs). To further refine the QTL regions and to identify candidate genes underlying seed oil fatty acid composition, we developed a chromosome level reference genome of Suneson, one of the parental varieties of the RILs. The reference genome was assembled de novo using Pacbio HiFi reads, scaffolded with HiC reads, gap filled using Pacbio CLR reads, and error corrected with highly accurate short read data from the Illumina HiSEQ platform. The chromosome and contig N50 is 31,294,119 bp with all 20 chromosomes and an additional 1602 contigs. Using RNAseq data from seven different plant tissues we produced a transcriptome with 138,304 total genes and 192,784 gene models. Using this new reference genome, we recalled SNPs of the resequenced germplasm lines and RILs. The previously identified QTL windows were narrowed significantly, and candidate genes were discovered. A particularly interesting QTL resides on chromosome 1 which contributes to fatty acid desaturation, i.e., oleic and linolenic acid contents. This region contains a gene encoding a transcription factor MYB67, which was also identified previously in Arabidopsis by GWAS for seed fatty acid composition. To further pinpoint candidate genes of oleic acid desaturation, we are analyzing transcriptomes of six lines of high and low oleic acids. 1.2 Phenotypic changes by heat stress Improving tolerance to high temperatures is essential for camelina agronomic sustainability. Two genotypes, Suneson and Pryzeth, were exposed to a transient 14-day heat stress at 37°C during the reproductive stages. Four cohorts of pods along the main stem, which were at different stages from fully developed pods (C1), young pods (C2), open flowers (C3) and flowering buds (C4) at the time of heat treatment, were examined for morphological and seed quality traits at maturity. The main stem length was shortened in both genotypes. Pods and seeds in all cohorts were negatively affected by heat, resulting in lower seed yield and reduced oil content. Seed size and seed weight had the greatest reduction in C1, pod size reduction was found the most in C3, and the number of fertile pods that contain at least one seed was reduced in C3 and C4. These results suggest that heat stress effects are developmental stage specific. Heat stress significantly reduced fertility during flowering and inhibited storage product biosynthesis and accumulation during seed filling which resulted in smaller and lighter seeds. Analyzing seed composition indicated that oil content decreased while protein content increased in seeds from heat treated plants. In addition, fatty acid composition was altered with the reduction of omega-3α-linolenic acidand concomitantly increased omega-6 linoleic acid being the most significantly affected. Our results also revealed the different responses in the two genotypes examined, suggesting genetic variation in camelina germplasm which can be explored to improve heat tolerance. 1.3 QTL and Transcriptomic analyses of reproductive development under heat treatments Elucidating the genetic response to high temperatures is essential for successful breeding of heat tolerant camelina varieties. Here we report a combinatorial approach to identifying candidate genes associated with heat stress by quantitative trait locus (QTL) mapping and comparative transcriptome profiling. A population of 257 recombinant inbred lines (RILs) was grown in a controlled growth chamber as described above under the high temperature regimes for 14 days beginning at the onset (with two open flowers) of the reproductive stage. Several traits related to seed production were evaluated at maturity. The QTL analysis identified several regions with co-located traits on chromosomes 8, 10, and 12. Two RILs with contrasting phenotypic responses to heat stress were chosen for gene expression profiling via RNA sequencing. Multiple pathways and genes were found to be strongly affected by heat stress, and many genes expressed differently between the two RILs. Several genes identified within the QTL regions were considered strong candidates that may control heat tolerance during reproduction in camelina. Objective 2: Discovering novel mechanisms controlling ALA accumulation in seed We characterized a mutant line (#3246) that contains an elevated level of ALA (nearly 43% 18:3) compared to its wildtype background (about 35% ALA). Quantitative PCR (QPCR) results indicated that the fatty acid desaturase (FAD3) gene was upregulated during seed development in the mutant. During the last reporting period, we conducted bulk segregant analysis (BSA) using F2 plants producing highest (>40% 18:3) and lowest (<35% 18:3) levels of ALA and identified a putative fragment in chromosome 10 that may contain the causal gene of the mutation. During this period, we have generated RNAseq data from developing seeds of the mutant and wildtype. Initial analysis of the transcriptomes resulted in many genes that are differentially expressed. Although some of those genes are known to be involved in fatty acid composition, e.g.,ABI3, further QTL mapping is needed to shortlist the candidate genes for molecular characterization. To validate the QTL result and further pinpoint candidate genes, we have conducted back-crossing with the wildtype plant Suneson and are choosing high and low ALA lines by half-seed fatty acid measurements for more precise bulk segregant analysis and deeper coverage (~40X) Illumina sequencing. Objective 3: Testing candidate genes QTL mapping experiments identified a few candidate genes on chromosome 1 that may contribute to oleic desaturation. We have obtained Arabidopsis T-DNA insertion mutants (e.g., MYB67) to test whether there is any effect on seed fatty acid composition. Primers for additional genes in that region have been designed for overexpression and/or CRISPR knockout experiments. Meanwhile, we will generate overexpression and CRISPR knockout mutations in camelina. Constructs are also being made for six other candidate genes (DGAT2, ABI3, MYB94 and GDSL1, 2, 6) and transgenic seeds have been obtained for two genes (GDSL6,DGAT2) in camelina.
Publications
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Progress 01/15/22 to 01/14/23
Outputs
Target Audience:Plant lipid biochemists and other plant scientists Biotechnology and oilseed businesses Members of the local community interested in agricultural research Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The funding has supported the work of one postdoc who is working on microRNA overexpression and the characterization of high-linolenic camelina mutants, and one graduate student who is working on the effect of high temperature during reproductive development on the accumulation of seed fatty acids and other related traits in camelina. How have the results been disseminated to communities of interest?Results were reported in a scientific conference (American Association of Industrial Crops 2022 annumal meeting)and departmental seminars. Manuscript will be prepared for publication in scientific journals during the next reporting period. What do you plan to do during the next reporting period to accomplish the goals?The next reporting period is the last one of this grant. We will therefore focus on the most promising results so far that may lead to publications. We will finish the mapping of QTLs for oil/fatty acid contents, and identification of candidate genes for heat responses during reproductive development. Candidate genes that are responsible for increased ALA in the EMS-mutants will also be identified. Verification of candidate genes will be assisted by comparative transcriptome analyses.We will then test the candidate genesby CRISPR gene knockouts or overexpressing studies.
Impacts
What was accomplished under these goals?
Major accomplishments of this reporting period are (1) the identification of QTLs and candidate genes related to fatty acid metabolism and heat responses, and (2) phenotypic changes of plant growth and seed characteristics in plants that are subject to heat treatment during reproductive stages. These results will not only guide our research for the next reporting period, but also may have significant impact on enhancing climate resilience of camelina for sustainable production of plant oils. Objective 1: Isolation of QTLs and candidate genes for fatty acid desaturation and heat tolerance in natural variations During the last reporting period, we have initiated the experiment to investigate the responses of camelina to high temperatures and the effect on the accumulation of ALA in seed.We designed an experiment involving a heat-stress period of two weeks beginning at the onset of flowering. Stress conditions included a transient daily heat stress with a high of 37ºC and a nightly recovery temperature of 21ºC. The genotypes tested were the lines MT5 and MT102. Here, we analyzed a broad range of phenotypes, including morphology, seed yield and oil contents. Most strikingly, the main stems of both genotypes were significantly impacted by heat stress. On average (n=6), main stems of MT5 decreased in length by 50% and by 38% in MT102. This was accompanied by a decrease in length between pods by approximately 30% in both genotypes. Deviating from expectations, seed size remained largely unchanged, with MT5 main stem seeds increasing in size by 2% and decreasing by 3% in MT102. Seed weight of side stems increased by 16% under heat stress in MT5 and decreased by 2% in MT102 on average. Average seed weight on main stems also increased in MT5 under heat stress, with an 11% increase on average. MT102 saw no change in seed weight between treatment groups. Total oil contents, quantified as a percentage of total seed mass, decreased by 5% in MT5 and 3% in MT102. Looking specifically at fatty acids, the amount of oleic acid (18:1) decreased in both genotypes (8% in MT5 and 18% in MT102). The amount of linoleic acid (18:2) decreased in both genotypes as well (12-13% for both genotypes). Interestingly, the amount of linolenic acid (18:3) increased by 13% in MT5 and remained unchanged in MT102. This highlights a key difference in genotypes, as the composition of fatty acids in response to heat stress is variable. The increasing or static average seed mass combined with lower oil contents indicate an increase in accumulation of other storage products. To identify candidate gene regions, we carried out a QTL mapping study using 257 recombinant inbred lines (RILs) derived from the parent lines MT5 and MT102. Six reps of the population were grown, with three being grown under control conditions and three being exposed to a two-week heat stress at the beginning of flowering. The heat stress profile was the same as above. Morphological traits for seeds and pods were collected, and fatty acids are currently being quantified. As expected, all of the key traits decreased on average across the population. Seeds per pod, seed weight, average seed area, and average pod area decreased by 32%, 8%, 3%, and 33% respectively. Using SNP data, we evaluated all of the collected traits to identify QTL regions. Two interesting QTL regions were identified. Mapping heat treated seed areas returned a significant region on chromosome 1. Close to the mapped SNP, two candidate genes, a predicted heat shock protein (HSP20) and a known heat shock-involved transcription factor (BZIP28) were found. Both genes have been previously shown to be upregulated under heat stress in Arabidopsis. Our second interesting QTL region of interest yielded the highest LOD score of all traits tested. The QTL peak mapped directly onto two genes, both aspartyl proteases. Less is known about this gene in oilseeds, as the gene family is large and has multiple functions. These candidate genes will be verified and characterized in future research. Objective 2: Discovering novel mechanisms controlling ALA accumulation in seed Approach 1: Characterization of mutants. A high-ALA (18:3) mutant line #3246 was crossed with its parent line Suneson for the bulked segregant analysis. F2 plants producing highest (>40% 18:3) and lowest (<35% 18:3) levels of ALA were pooled to generate genome sequences by Illumina sequencing. We compared those sequences with the Suneson reference genome that we assembled recently. Using the MutMap approach, we identifieda fragment in chromosome 10 starting from 1123172 to 2037143 that may carry the high-ALA mutation. Since this is a large region (913,971bp) that contains 224 SNPs, we will perform further analysis using the Cyverse pipeline. Also, we will generate RNAseq data from the mutant and wildtype to narrow down this QTL region. As we are finishing annotating the reference genome (Suneson), we will identify the candidate genes to test their roles in ALA accumulation using approaches in Objective 3. Approach 2: MicroRNA misexpression.We have generated a total of 82 miRNA transformed plants varying 22 miRNA vectors. Our preliminary results indicated that fatty acid composition was only moderately affected (increase/decrease by >5% of 18:3 compared with the WT controls) in the seeds: Overexpressing miRNAs (miR857, miR400 and miR86018:3) caused decreased 18:3, while others (miR11139, miR165, miR408, miR168, miR173 and miR162) increased 18:3.Twelve more plants which also had changed 18:3 content need to be determined for their miRNAs. These plants will be further analyzed in their next generations to determine whether the above-mentioned miRNAs truly affect fatty acid metabolism in camelina seeds. Objective 3: Testing candidate genes Low efficiencies of gene editing were noted in our recent transgenic experiments. We have refined our CRISPR/Cas9 procedures to facilitate the testing of candidate genes for 18:3 accumulation and heat tolerance that will be identified in Objectives 1 and 2. This involved the incorporation of multiplexed guide RNAs to target the candidate genes more effectively for mutagenesis. We are in the process of making the constructs for HSP20 and bZIP28.
Publications
|
Progress 01/15/21 to 01/14/22
Outputs
Target Audience:Plant lipid biochemists and other plant scientists Biotechnology and oilseed businesses Members of the local community interested in agricultural research Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The funding has supported the work of one postdocwho is working on microRNA overexpression and the characterization of high-linolenic camelina mutants,and one graduate student who is workingon the effect of high temperature during reproductive development on the accumulation of seed fatty acids and other related traits in camelina. How have the results been disseminated to communities of interest?One article has been published describing the characterization of camelina mapping piopulations including a diversity panel of natural germplasms and a recombinant inbred lines derived from a cross between two contrasting varieties. Candidate genes affecting fatty acid composition in camelina seed were identified or discussed. What do you plan to do during the next reporting period to accomplish the goals?The plan for the next period is largely based on the origional proposal. The following briefly describes the work in 2022: Objective 1: Mapping QTLs and candidate genes As we will have the high-quality genome of camelina (cv. Suneson) assembled, we will recall the SNPs in the natural mapping populations (diversity accessions and the recombinant inbred lines). We expect that the QTL regions can be refined by the high quality markers and the genome. New phenotypic data will also be obtained by growing plants under high temperature conditions. Objective 2: Discovery of novel genes/genetic mechanisms of linolenic acid accumulation More transgenic plants expressing 35S-miRNAs will be generated and characterized. Candidate genes in the high-ALA mutants will also be identified by the MutMap approach. We will conduct genome resequencing of contrasting (high and low ALA in seeds) phenotypesand compare with the reference genome of Suneson. Objective 3: Functional testing of candidate genes We will complete the characterization of DGAT2 CRISPR and overexpressing lines. Newly identified candidate genes from Obj. 1&2 will be tested using similar approaches.
Impacts
What was accomplished under these goals?
The project was affected by the restrictionsrelated to COVID-19 during the first few months.Apostdoc arrived 4 months after the project start date, and a graduate student joined the research group in August, 2021. Nevertheless, the project is right on track to suceed and we have made the following progress during the first period of the grant: Objective 1: Isolation of QTLs and candidate genes for fatty acid desaturation in natural variations We evaluated genetic variation among a worldwide collection of 222 Camelina sativa accessions. Before the inception of this project,we performed whole-genome resequencing to obtain single nucleotide polymorphism (SNP) markers and to analyze genomic diversity. We also conducted field evaluations in two consecutive seasons for variations in key agronomic traits related to oilseed production such as seed size, oil content (OC), fatty acid composition, and flowering time. We determined the population structure of the camelina accessions using 161,301 SNPs. Further, we identified quantitative trait loci (QTLs) and candidate genes controlling the above field-evaluated traits by genome-wide association studies (GWAS) complemented with linkage mapping using a recombinant inbred line (RIL) population. These results have been published and the NIFA support was acknowledged. Particularly,we mapped QTLs affecting seed fatty acid composition in the camelina genome. A FAD2 locus was found to explain the variation of unsaturated fatty acid levels in the natural population. The genetic artitecture of the a-linolenic (ALA) levels in seed is complex, however, we identified a few candidate genes including a diacylglycerol acyltransferase 2(DGAT2), which we will verify and test their functions in Obj. 3. In addition, we have intiated the experiment to investigate the responses of camelina to high temperatures and the effect on the accumulation of ALA in seed.The parents of the RIL population, Suneson and Prytzh, were grown in a growth chamber. After flowering, the temperature was gradually raised from 25°C to 35°C and stayed at 35°C for 4 hours/day for vaious durations (3, 7 and 14 days). We are assessing the effects of high temperatures on seed characteristics including oil content and fatty acid composition. Oncethe temperature treatment regime is determined that creats the profound effect on ALA level, we will treat the mapping populations to map QTLs and candidate genes. Objective 2: Discovering novel mechanisms controlling ALA accumulation in seed Approach 1: Characterization of mutants. EMS mutants of high ALA in seed have been crossed with their wildtype parent, Suneson. To map the causal genes in the mutants using the MutMap approach, we have obtained the genome sequence of Suneson generated by the latest PacBio sequencing technology. A reference genome is being assembled. Our preliminary results indicate that the genome quality is high and is significantly improved over the previously published genome (Kagale, et al., 2014). We expect that the improved reference genome will enhance the power to dected mutant alleles in the high-ALA lines, which we will follow up with functional determinations. Approach 2: MicroRNA misexpression. To discover novel mechanisms controlling fatty acid modification in camelina seed by microRNA misexpression, we have generated 30 constructs of miRNAs driven by the 35S promoter. These constructs have been transformed into camelina plants. We have confirmedthat 13 miRNAs have been successfully transformed into camelina plants. More transgenic are being generated, and the effects of these miRNAs on seed fatty acid composition will be analyzed using gas chromatography. We will also observe and characterize other possible growth and development traits induced by miRNA misexpression. Objective 3: Testing candidate genes We are testing candidate gens using CRISPR and seed-specific overexpression in transgenic plants. Constructs of CRISPR targeting two regions in the DGAT2 genes, and overexpression by seed specific phaseolin promoter have been transformed into the camelina Suneson plants. Tansgenic plants (40 CRISPR, 31 over-exprsssing) have been obtained. We are detecting the RISPR-induced mutation sites.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Li, H., Hu, X., Lovell, J. T., Grabowski, P. P., Mamidi, S., Chen, C., Amirebrahimi, M., Kahanda, I., Mumey, B., Barry, K., Kudrna, D., Schmutz, J., Lachowiec, J., & Lu, C. (2021). Genetic dissection of natural variation in oilseed traits of camelina by whole-genome resequencing and QTL mapping. Plant Genome, 14 (2), e20110. https://doi.org/10.1002/tpg2.20110
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