Progress 09/01/23 to 08/31/24
Outputs
Target Audience:The target audience is sorghum researchers at government agencies, universities and commercial companies as well as government regulatory agencies.? Changes/Problems:Days to flowering measurements determined under greenhouse conditions are incongruent with ones determined in the field. Therefore, all critical data to the project on days to flowering will be determined in the field, which has limited the data collection windows. The data from 2023 field season was completely lost due to drought conditions at the dry land field site near Lincoln, NE. What opportunities for training and professional development has the project provided?A second post-doctoral research associate was hired for this project in March 2024, the training of this individual consists of informal training through research seminars, discussions on scientific literature and weekly meetings with the PD and co-PI of the project. How have the results been disseminated to communities of interest?This project was included in a presentation at the Sorghum Improvement Conference of North America (SICNA) in March at Oklahoma City, OK. In addition, this project was included in a poster presentation at Gordon Research Conference on Lignin in July at Easton, MA. In addition, this project was presented at the project directors' meeting in May. What do you plan to do during the next reporting period to accomplish the goals?Objective 2: The F2 progeny from EBA-3 combined with Hegari (Ma4) and Early Hegari (ma4) will be evaluated in the field for flowering time to identify lines flower than EBA-3. Objective 3: AgriPlex genotyping platform will be used identify the alleles from EBA-3 by Hegari (Ma4) and Early Hegari (ma4) F2 progeny using from Objective 2. Objective 4: Two transgenic lines will be crossed with EBA-3, A3 and A4 CMS lines to evaluate these containment strategies (sterility and non-flowering) under field conditions.
Impacts
What was accomplished under these goals?
Objective 1: to evaluate the efficacy of A3 and A4 cytoplasmic male sterility (CMS), the two year replicated trial was completed this fall where seed set under pollination bags, which examined as the component of fertility restoration of A3 and A4 CMS hybrids with Tx430. None of four A4 CMS hybrids combination displayed any seeds in all four replications at the Mead, NE location. Only the hybrid, A3 Redland X Tx430 displayed low fertility restoration activity of 58 A3 CMS hybrids combinations evaluated in replication at the Mead location. Fewer than 10 seeds per 6 panicles were observed in this low fertility interaction. The fertility was not able to be evaluated at the dry land location near Lincoln, NE due drought in 2023. This information will be compared with the pollen staining data, but preliminary analyses indicated that A3 and A4 cytoplasmic male sterility are reliable means to prevent pollen flow from transgenic sorghum. Objective 2, to develop a sorghum line that complements the Tx430 Ma alleles, the sorghum maturity line series 44M, 58M, 60M, 80M, 90M and 100M crossed to A3Tx430 and the flowering time was evaluated in the field in 2024. The hybrids with 80M (Ma3) flowered earlier than the ones with 60M (ma3), which indicate the Ma3 locus had a relatively minor effect on flowering in our environment. Objective 3: to determine the functionality at Ma4 locus inTx430 and EBA-3, these lines were cross-pollinated with Hegari (Ma4) and Early Hegari (ma4). The progeny were grown in the Mead and Lincoln, NE field locations in summer 2024. EBA-3 combination with both Hegari (Ma4) and Early Hegari (ma4) failed to reach 50% anthesis at both field locations. Thus, it was impossible to determine whether EBA-3 contain a recessive, loss of function allele or a functional allele at the Ma4 locus. In contrast, Hegari (Ma4) and Early Hegari (ma4) in combination with Tx430 reached 50% anthesis at 242 and 235 days (Julian calendar days), which indicated ma4 alleles contribute toward flowering 7 days earlier. These data involving the combination with EBA-3 and Tx430 suggest the alleles at Ma4 locus have a relatively small impact on the timing of flowering. This observation is similar to 80M (Ma3) and 60M (ma3) in Objective 2. Thus, efforts to examine alleles at these loci will be focused on the other four Ma loci, Ma1, Ma2, Ma5 and Ma6.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Grant, N. P., Toy, J. J., Funnell-Harris, D. L., & Sattler, S. E. (2023). Deleterious mutations predicted in the sorghum (Sorghum bicolor) Maturity (Ma) and Dwarf (Dw) genes from whole-genome resequencing. Scientific Reports, 13(1), 16638. doi:10.1038/s41598-023-42306-8
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Progress 09/01/22 to 08/31/23
Outputs
Target Audience:The target audience is sorghum researchers at government agencies, universities and commercial companies as well as government regulatory agencies.? Changes/Problems:Days to flowering measurements determined under greenhouse conditions are incongruent with ones determined in the field. Therefore, all critical data to the project on days to flowering will be determined in the field, which will limit the data collection windows.? What opportunities for training and professional development has the project provided?A post-doctoral research associate was hired for this project in June 2021, the training of this individual continued in 2nd year of the project, which end in June 2023 with the candidate taking another post-doctoral training position. This individual utilized several on-line courses to further his training in bioinformatics analyses. This post-doctoral researcher also participated in USDA- ARS post-doctoral professional development network to enhance their training. How have the results been disseminated to communities of interest?A poster presentation was given at the Plant and Animal Genome Conference (PAG30) in January 2023. A peer-review manuscript was submitted and accepted for publication in Scientific Reports, which characterized the Ma alleles from 880 re-sequence sorghum accessions recently made available to the public. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: A handful A3 and A4 hybrids produced pollen that stained with positive for viability without restoring seed set, which will be investigated further in the next reporting period. Objective 2: The F2 progeny from Tx430 and EBA-3 combined with Hegari (Ma4) and Early Hegari (ma4) also will be evaluated in the field for flowering time. Objective 3: Plans to identify the alleles whose AgriPlex markers failed will be developed. Analyze data from targeted genotyping to determine Ma alleles of A3 and A4 genetic stocks, which will identify the most appropriate combinations between the cytoplasmic male sterility and function Ma sorghum genetic stocks. F2 progeny of EBA-3 X Early Hegari screen for ma2, ma3R, ma5, and Ma6 in the field. Objective 4: Cross EBA-3 containing A3 and A4 CMS with transgenic lines to determine whether both strategies prevent pollen flow from the progeny via sterility or non-flowering.
Impacts
What was accomplished under these goals?
Objective 1: to evaluate the efficacy of A3 and A4 cytoplasmic male sterility (CMS), the pollen from progeny of 58 A3 and 4 A4 CMS lines cross-pollinated by Tx430 were iodine stained and visualized under the microscope to determine if the CMS trait prevented production of viable pollen in all Tx430 hybrids. The staining results indicated that 27 of 225 samples contained high-levels of viable pollen, which may indicate that A3 and 4 A4 CMS may not resulted in sterility across all genotypes. This information will be compared with the two year replicated trial completed this fall where seed set under pollination bags was examined as the component of fertility restoration of A3 and A4 CMS hybrids with Tx430. Together these data once analyzed will determine whether A3 and A4 cytoplasmic male sterility are reliable means to prevent pollen flow from transgenic sorghum. Plant height, flowering date, and seed set under pollination bags were recorded from field and greenhouse grown plants for Objectives 2 and 3. However, flowering dates between the greenhouse and field were incongruent, thus for the remainder of the project our efforts will be focused on field results. Objective 2: to determine the functionality at Ma4 locus inTx430 and EBA-3, these lines were cross-pollinated with Hegari (Ma4) and Early Hegari (ma4). The progeny were grown in a growth chamber under a short-day photoperiod (10-hour day/14-hour night) to induce flowering. However, the plants did not flower under these conditions, thus this experiment will be repeated in the field next summer to determine the Ma4 alleles of EBA-3 and Tx430. A3 Tx430 X EBA-3 hybrids were evaluated in in the field to determine flowering time, which did not flower in the field and demonstrated EBA-3 combined with Tx430 will prevent pollen flow. Objective 3, to determine Ma alleles for 58 A3 and 4 A4 CMS lines, DNA was extracted from germinated seedlings. The AgriPlex platform was used to determine alleles at the Ma loci. Most alleles were successfully interrogated using the marker developed by AgriPlex. This information will be used to select the most appropriate sources of CMS to combine with complementary Ma alleles to restore photoperiod sensitivity and prevent viable pollen production. Most Ma alleles of Tx430 and EBA-3 were confirmed using the AgriPlex platform in Objective 2. Objectives 2 and 3: To utilize newly published information and complement the planned experiments in these objectives, 880 sorghum accessions were sequenced using next-gen technologies and these data were made publicly available through two publications (Lozano et al., 2021 and Boatwright et al., 2022). The raw sequencing reads for 5 characterized Ma and 3 Dwarfing (Dw) genes were analyzed to identify potential loss of function alleles at these loci, which was accepted for publication.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Grant, N. P., Toy, J. J., Funnell-Harris, D. L. and Sattler, S. E. 2023. Deleterious mutations predicted in the sorghum (Sorghum bicolor) Maturity (Ma) and Dwarf (Dw) genes from whole-genome resequencing. Scientific Reports 13: 16638 10.1038/s41598-023-42306-8
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Progress 09/01/21 to 08/31/22
Outputs
Target Audience:The target audience is sorghum researchers at government agencies, universities and commercial companies as well as government regulatory agencies.? Changes/Problems:The AgriPlex Genomics platform will be utilized to genotype the Ma loci in place of next-gen sequencing platforms mentioned in the grant proposal for Objectives 2 and 3. What opportunities for training and professional development has the project provided?A post-doctoral research associate was hired for this project in June 2021, the training of this individual continued in 2nd year of the project. This individual has utilized several on-line courses to further his training in bioinformatics analyses. This post-doctoral researcher has also participated in USDA- ARS post-doctoral professional development network to enhance his training. How have the results been disseminated to communities of interest?Two presentations on-line were given on this project during this year. A peer-review manuscript is also in preparation, which characterizes the Ma alleles from 880 re-sequence sorghum accessions recently made available to the public. What do you plan to do during the next reporting period to accomplish the goals?Objective 2: Ma alleles of Tx430 and EBA-3 will be confirmed using the AgriPlex platform. Progeny from cross-pollination between EBA-3 and A3 Tx430 will be evaluated in the field for flowering time. The F2 progeny from Tx430 and EBA-3 combined with Hegari (Ma4) and Early Hegari (ma4) also will be evaluated in the field for flowering time. These experiments will determine the effects on flowering time of Ma/ma constituents at the six loci and demonstrate effects of these lines when combined together. EBA-3 cross-pollinations introduce the Ma6 allele into germplasm with Ma1 to develop a female line when combine with Tx430 prevents flowering under a temperate photoperiod. Objective 3: Analyze data from AgriPlex targeted genotyping to determine Ma alleles of A3 and A4 genetic stocks, which will identify the most appropriate combinations between the cytoplasmic male sterility and function Ma sorghum genetic stocks.
Impacts
What was accomplished under these goals?
Objective 1: to evaluate the efficacy of A3 and A4 cytoplasmic male sterility (CMS), pollen was collected from progeny of 58 A3 and 4 A4 CMS lines cross-pollinated by Tx430 to determine if the CMS trait prevented production of viable pollen in all Tx430 hybrids. Plant height, flowering date, and seed set were recorded from greenhouse grown plants for Objectives 2 and 3. Microscopy using iodine staining was used to examine the loss of pollen viability in the progeny. This experiment was followed-up with a replicated field study grown this summer where the seed set was measured from the progeny of these crosses. Together these data once analyzed will determine whether A3 and A4 cytoplasmic male sterility are reliable means to prevent pollen flow from transgenic sorghum. Objective 2: to determine the functionality at Ma4 locus inTx430 and EBA-3, these lines were cross-pollinated with Hegari (Ma4) and Early Hegari (ma4). The progeny were grown in a growth chamber under a short-day photoperiod (10-hour day/14-hour night) to induce flowering. Objective 3, to determine Ma alleles for 58 A3 and 4 A4 CMS lines, DNA was extracted from germinated seedlings. The AgriPlex platform will be used to genotype Ma loci in the next funding period. This information will be used to select the most appropriate sources of CMS to combine with complementary Ma alleles to restore photoperiod sensitivity and prevent viable pollen production. Objectives 2 and 3: To utilize newly published information and complement the planned experiments in these objectives, 880 sorghum accessions were sequenced using next-gen technologies and these data were made publicly available through two publications (Lozano et al., 2021 and Boatwright et al., 2022). The raw sequencing reads for 5 characterized Ma and 3 Dwarfing (Dw) genes were analyzed to identify potential loss of function alleles at these loci, which would be useful for this project and other sorghum breeding endeavors.
Publications
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Progress 09/01/20 to 08/31/21
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A post-doctoral research associate was recruited and hired for this project in June 2021. During the COVID-19 pandemic, this individual has utilized several on-line courses to further his training in 'big data' analyses and bioinformatics. This post-doctoral researcher has also participated in USDA- ARS post-doctoral professional development network to enhance his training. 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?Objective 1: In field, the A3 and A4 CMS hybrids between CMS lines as the female parent and Tx430 as the pollen source will be evaluate in the summer of 2022. The experiment will determine whether A3 or A4 CMS completely blocks male fertility in combination with Tx430, a critical way to prevent pollen flow. Objective 2: Pollination between 'Hegari' and Tx430 will determine whether Tx430 has a functional Ma4 allele, which is critical information to develop a line that when combine with Tx430 prevents flowering under a temperate photoperiod. EBA-3 cross-pollinations introduce the Ma6 allele into germplasm with Ma1 to develop a female line when combine with Tx430 prevents flowering under a temperate photoperiod. Objective 3 crosses A3 and A4 CMS sources will be cross-pollinated with EBA-3 to determine whether there are any unexpected consequences when combining the Ma6 source (EBA-3) and either CMS system to prevent pollen flow in sorghum.
Impacts
What was accomplished under these goals?
Cytoplasmic male-sterility (CMS) is one way to prevent pollen-flow from transgenic plants. Several CMS sources have been identified in sorghum, but A3 and A4 cytoplasmic male sterility have a very limited number of known restorer lines and these lines do not restore full fertility. Objective 1: Previously developed 58 sorghum parental lines containing A3 and four lines containing A4 CMS were grown in the greenhouse in 2020-2021, and these lines were cross pollenated by RTx430. The resultant hybrid seed will be planted in field locations in 2022, where fertility of the F1 plants will be evaluated. These experiments will determine whether A3 or A4 CMS sources can block male fertility in Tx430 F1 hybrids. Sorghum was converted from a short-day flowering plant to a photoperiod insensitive one using spontaneous loss of function mutations at six loci (Maturity; Ma1 to Ma6), and different combinations of functional and non-functional alleles at these loci allow this crop to flower under long-day photoperiods (>11 hours). Objective 2: Hybrids containing at least five out of six functional alleles (Ma1--Ma2--Ma3--Ma4--Ma5--Ma6--) are expected to flower extremely late under a temperate photoperiod (approximately 120 days), and this strategy was successfully used to develop photoperiod sensitive forage/energy sorghum. Our goal is to complement ma1 and ma6 alleles of Tx430, which is expected to result in F1 hybrids that would flower after 120 days instead of 70 days required for Tx430 to flower under a temperate photoperiod. Genome sequences for 14 lines: RTx430, N98, N100, N108, N109, N110, 'Early Hegari', Tx2737, BTx623, 'Brawley', 'Greenleaf', 'Wray', RT7000, and 'Sugar Drip' were identified in public databases, which are being used to determine whether these lines have functional alleles at Ma1, Ma2, Ma3, Ma5 and Ma6 as well as height controlling loci Dw1 Dw2 and Dw3. The 62 and 21 lines containing A3 and A4 CMS, respectively were grown in the greenhouse for DNA extraction to confirm Ma allelic constituents using PCR based markers. EBA-3, one of the only sources with a functional Ma6 allele, was grown in the greenhouse, and cross pollinations between it and several lines were initiated. Together, these experiments will identify the combination of alleles necessary to prevent flowering under a temperate photoperiod in transgenic Tx430 F1 hybrids.
Publications
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