Progress 06/01/24 to 05/31/25
Outputs
Target Audience:Growers, Scientists, and administrators from various farmersorganizations.? Efforts: One of our manuscripts was recently accepted for publication in the Plant Disease Journal (https://doi.org/10.1094/PDIS-10-23-2176-RE). Additionally, we delivered oral presentations at regional scientific meetings (Western American Society of Plant Biologists) and the United States Dry Peas, Lentils, and Pulse annual grower meeting. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One undergraduate student was trained in this project, where they gained hands-on training on controlled condition phenotyping protocols.? How have the results been disseminated to communities of interest?Two oral presentations were delivered at the USAPulses and NIFA project awardee annual meeting to update different stakeholders about the progress made in the project.? What do you plan to do during the next reporting period to accomplish the goals?We will complete cloning of theC-Ph1gene oligo into the TRV2-based VIGS vector to accomplish alien gene transfer approach using the F1s developed betweenC. arietinumandC. bijugum. Additionally, we will make additional crosses betweenC. arietinumandC. reticulatumaccessions and will screen them to select first-generation ascochyta blight resistant germplasm. In the coming planting season, we will be field screening the wild germplasm of chickpeas. Similarly, we will be developing and validating the PCR-based markers for ascochyta blight identified from the GWAS mapping. As an additional activity, we will functionally validate the candidate genes mapped using the GWAS study for their role in Ascochyta blight resistance. A detailed gene expression analysis for the whole gene family is currently underway, and we are also in the process of validating the gene's functionally using the RNAi-based gene silencing and gene overexpression inC. arietinumsusceptible genotypes. We will continue with these additional activities.
Impacts
What was accomplished under these goals?
Thegoalof the project isto develop a simple, targeted, precise and fast method of alien gene transfer in chickpeas (Cicerarietinum)and use it to transfer a high level of resistance toAscochyta blightcaused byAscochyta rabiei.. Thespecific objectivesof the project are: Identify and confirm novel sources of Ascochyta blightresistance among the wild species, Develop and optimize a novel alien gene transfer approach in chickpea, Develop breeder-friendly markers for the alien gene(s) controlling Ascochyta blightresistance, and Field trials to confirm Ascochyta blightresistance and other agronomic traits among develop germplasm. Answer: Objective#1: Identify and confirm novel sources of Ascochyta blightresistance among the wild species: A previously described mini-dome assay and our new controlled condition screening protocol were used for the ascochyta blight screening(Chenet al., 2005).Briefly, 14 days after planting, chickpea seedlings were inoculated with a pycnidia spore suspension at a concentration of 2x106spores/ml from a 7-day-old culture of a local isolate of theAscochyta rabieicollected from the field-grown chickpea seeds in Pullman, WA, as previously described(Chenet al., 2005). About 2 ml of the spore suspension was sprayed per plant per accession. After inoculations, plants were placed on a platform inside a plastic container containing water and covered using a plastic sheet for 48 hours. Disease reaction was scored 21 days post inoculations using a modified previously defined 1-9 disease scoring scale(Chenet al., 2005). Briefly, 1- healthy plant, no disease; 2- lesions present, but small and inconspicuous; 3- lesions visible, but restricted; 4- clearly visible severe lesions; 5- stem girdling due to lesions, most leaves show lesions; 6- plant collapsing, tips die back; 7- plant dying, but at least three green leaves visible; 8- virtually dead plant (no green leaves left), but with green stem; 9- dead plant. The mean disease score of individual accessions was used for statistical analysis. Selected resistant accessions from our previous work further screened against two additional isolates ofAscochyta rabieicollected from Washington and Montana under controlled conditions and compared with the most commonly used source of partial resistance from cultivated chickpeas (ICC 12004). This is compared toC. arietinumaccession, whose mean disease score rating was 5.3, 6.7, and 6.7 for isolates from Montana, Idaho, and Montana, respectively (Figure 1). Whereas, for mean disease rating for four accessions (W6 51172, W6 51175, W6 51378, and W6 51375) revealed a mean disease score between 1.1-2.2 (Figure 1). Additionally, about 33 accessions were screened against a pure isolate of a highly virulent strain ofAscochyta rabiei(AR628) using controlled condition screening. In comparison to the susceptible control (Spanish White) that showed a disease resistance score of 6.8, two accessions from the wild germplasm were identified with a mean disease score of 1.75 and 2, which was significantly lower than the known source (PI 12004) of genetic resistance used in chickpea breeding. A similar field-based screening of the 110 wild chickpea accessions was performed in Montana and Idaho in replicated 10 ft long rows. Disease scoring was done before and after flowering on a 1-9 scale, as detailed above. No artificial inoculum was used, but a susceptible genotype (Spanish White) was used as a spreader. No disease was observed in Idaho under natural conditions, whereas the disease was present in Montana during 2023. As expected under field conditions, the disease spread was non-uniform, as evident from the disease rating of the partially resistant cultivated chickpea genotypes, i.e., Billybeans and Sawyer. Billybeans had a mean disease score of 1, whereas Swayer had a mean disease score of 2.5. Out of the 110 accessions tested under field conditions, about 67 had a mean disease score of less than 3, and 10 accessions had a mean disease score of more than 5 (Figure 2). Objective#2: Develop and optimize a novel alien gene transfer approach in chickpeas:The optimization of the alien gene transfer method is reliant on the availability of an efficient virus-induced gene silencing system in chickpeas. To develop the F1s between cultivated and wild chickpeas, we have optimized the embryo rescue and regeneration protocols. Reciprocal crosses between cultivated chickpeas (Swayer and Billybeans) and wild chickpeas (Cicer reticulatumandC. bijugum) were attempted. About 200 flowers were pollinated with Swayer, which yielded no viable F1 pods or plants, whereas, with Billybeans as the male parents, about 20 flowers ofC. bijugumwere pollinated, which resulted in 10 cross pods. The pods were treated with hormonal mixture (GA3 & IAA) to ensure pod viability. Additionally, about 40 flowers ofC. reticulatumwere pollinated with Billybeans as the male parent and female parents, respectively. Post and Pre-fertilization barriers were observed while usingC. reticulatumas the female parent, whereas successful hybridization was observed withC. reticulatumas the male parent and Billybeans as the female parent. Virus-induced gene silencing was optimized in chickpeas using the Tobacco mosaic virus (TMV) with thephytoene saturasegene as the phenotypic marker. Additionally, to test if the virus-induced gene silencing can be used to silence meiotic genes, we have cloned the DMC1 gene (known meiotic pairing regulator) to the TMV2 genome of the TMV-based gene silencing system. Objective#3: Develop breeder-friendly markers for the alien gene(s) controlling Ascochyta blightresistance:To further characterize the candidate genes for their role in ascochyta blight resistance, we are performing a time-lapse gene expression analysis study. Tolerant and susceptible accessions of theC. reticulatumwere planted and treated with three different isolates ofAscochyta rabieiand samples for RNA extraction were collected at 24, 48, 72, 96, and 120 hrs after infection. Total RNA was extracted using the hot phenol method followed by first-strand cDNA synthesis. Gene-specific primers for the DNA helicase gene family were designed after identifying and isolating all members of the SNF2 DNA helicase gene family using a bioinformatics-based pipeline and Primer-Blast program of NCBI to characterize the role of GWAS-based candidate genes for their role in Ascochyta blight resistance. ResistantC. reticulatumaccession carried a higher relative transcript abundance ofCr_14189.1andCr_14190.1compared to the susceptible accession (Figure 4).TheCr_14189.1transcript abundance was ~110-fold higher in resistant accession than in susceptible accession. Similarly, theCr_14190.1transcript was ~3-fold higher in resistant accession compared to susceptible accession. Objective#4: Field trials to confirm Ascochyta blightresistance and other agronomic traits among developing germplasm:110 accessions of wild chickpea germplasm were screened for ascochyta blight under field conditions in Bozeman, Montana. The germplasm was planted in nonreplicated rows of 10ft with spreader rows (Billy beans and Spanish White). All 110 germplasm accessions were hand-harvested and will be screened in the next planting season in replicated trials for ascochyta blight resistance across three locations.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2025
Citation:
Rawale KS, Gutierrez-Zamora GR, Venditto NA, Gill KS. Identification of Pathogen-Specific Novel Sources of Genetic Resistance Against Ascochyta Blight and Identification of Their Underlying Genetic Control. Plant Dis. 2024 Aug;108(8):2367-2375. doi: 10.1094/PDIS-10-23-2176-RE. Epub 2024 Aug 5. PMID: 38332491.
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Progress 06/01/23 to 05/31/24
Outputs
Target Audience:Growers, Scientists, and administrators from farmer's cooperatives. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Yes, we have presented our work at scientific meetings and annual grower meetings to update the scientific community and chickpea growers about the progress being made in the project. What do you plan to do during the next reporting period to accomplish the goals?This year, we are planning to publish our findings on the controlled condition screening of the wild germplasm using a local isolate. Additionally, we are further looking to screen the germplasm against the isolates collected from Montana to identify accessions carrying resistance against multiple pathotypes. Furthermore, we are currently developing breeding and genetic mapping populations to initiate the transfer of alien genes carrying ascochyta blight resistance to cultivated chickpeas. We are further developing breeder-friendly markers for the candidate genes identified for ascochyta blight resistance, which will be validated on the mapping population that are currently developing.
Impacts
What was accomplished under these goals?
Thegoalof the project isto develop a simple, targeted, precise and fast method of alien gene transfer in chickpeas (Cicerarietinum)and use it to transfer a high level of resistance toAscochyta blightcaused byAscochyta rabiei.. Thespecific objectivesof the project are: 1. Identify and confirm novel sources of Ascochyta blightresistance among the wild species, 2. Develop and optimize a novel alien gene transfer approach in chickpea, 3. Develop breeder-friendly markers for the alien gene(s) controlling Ascochyta blightresistance, and 4. Field trials to confirm Ascochyta blightresistance and other agronomic traits among develop germplasm. Answer: Objective#1: Identify and confirm novel sources of Ascochyta blightresistance among the wild species: We have screened 200 accessions of wild relatives of chickpeas from 8 Cicer species for ascochyta blight resistance against a local isolate selected and isolated from Genesee Idaho. Based on the controlled condition effort, we have identified ~25 accessions that confer a high level of resistance against a highly virulent isolate of ascochyta rabiei. Additionally, we collected isolates from Bozeman, Montana, that were purified at Geneshifters. Furthermore, we have screened 6 accessions showing high levels of ascochyta blight resistance against 3 isolates of ascochyta rabiei collected from Washington, Idaho, and Montana. Furthermore, field condition-based screening of the germplasm in Idaho and Montana yielded significant disease reactions in Montana before flowering and podding. After combining the data from controlled and field condition-based screening, we identified highly resistant accessions that carry both seedling and adult plant resistance. Objective#2: Develop and optimize a novel alien gene transfer approach in chickpeas: To develop the F1s between cultivated and wild chickpeas, we have optimized the embryo rescue and regeneration protocols. Previously, we had crossed cultivated chickpeas with C. pinnatifidum and performed embryo rescue using previously published protocols. However, the F1 embryos didn't germinate on the growth media. Since then, we have optimized the growth media recipe, and we are currently developing the F1s between Swayer and wild ascochyta blight-resistant accessions. Objective#3: Develop breeder-friendly markers for the alien gene(s) controlling Ascochyta blightresistance: To develop breeder-friendly markers, we performed a genome-wide association analysis in a subset of wild relatives screening under the controlled condition screening. Using the publicly available restriction site-associated sequencing (RAD-seq) raw sequence data and SNP call file (von Wettberg et al., 2018; Newman et al., 2021), we filtered the SNP data for 72 accessions used in our screening and performed a GWAS analysis. The mean disease reaction score was used as phenotypic data for GWAS analysis. The kinship matrix for the 72 accessions was developed using TASSEL (Bradbury et al., 2007), and the GAPIT3 package on R was run to identify genome-wide association using a multi-locus mixed linear model (Wang & Zhang, 2021). Using a stringent threshold, a single significant marker on chromosome 5 was identified for ascochyta blight resistance, which explained ~58% of the phenotypic variance for the trait (Figure 3). Additional comparison with the previously identified ascochyta blight-resistant SNPs from the same panel and previous studies revealed that the identified SNP against local Palouse isolate was unique. The associated SNP containing read mapped in the Cr_14190.1_v2 gene, which will be referred to as a candidate for the resistance. An additional gene (Cr_14189.1_v2) was identified in a 20kb region surrounding the candidate gene with a putative role in biotic stress, as described previously (Xie et al., 2018). We are currently sequencing the full-length candidate gene from our wild relatives to design breeder-friendly markers. Objective#4: Field trials to confirm Ascochyta blightresistance and other agronomic traits among developing germplasm: 110 accessions of wild chickpea germplasm were screened for ascochyta blight under field conditions in Bozeman, Montana. 110 accessions of wild chickpea germplasm were screened for ascochyta blight under field conditions in Bozeman, Montana. 110 accessions of wild chickpea germplasm were used for seed multiplication and disease screening at Pullman, Washington. Due to low seed availability, the germplasm was planted in nonreplicated rows of 10ft with spreader rows (Billy beans and Spanish White). All 110 accessions of the germplasm were hand-harvested and will be screened in the next planting season in replicated trails for ascochyta blight resistance across three locations.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2024
Citation:
Kanwardeep S. Rawale, Gemini R. Gutierrez-Zamora, Noah A. Venditto, and Kulvinder S. Gill
Identification of Pathogen-Specific Novel Sources of Genetic Resistance Against Ascochyta Blight and Identification of Their Underlying Genetic Control. https://doi.org/10.1094/PDIS-10-23-2176-RE
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Progress 06/01/22 to 05/31/23
Outputs
Target Audience:The target audience reached by our efforts include growers from three different states, scientific community, various administrators, farmer co-ops, and other stakeholders. The target audience was reached via oral presentation to various stakeholder's groups,annual meeting organized by USA Pulses, presentation at scientific meetings, one-on-one communication with various stakeholders, and social media outlets. Changes/Problems:Germination of the wild relative species seed, both in the greenhouse as well as in the field, was a big problem for the seed obtained from the gene bank thus the number of plants evaluated was less than intended. Additional plants will be planted this year and additional seed treatment will be used to increase the number of plants evaluated for each accession. No change is expected and this problem is not expected to have any negative impact on the project as the screening results were clear thus the selection of the desired genotypes was reliable. What opportunities for training and professional development has the project provided?The PI and the project administrator attended the annual ASA-CSSA-SSSA meeting at Baltimore, MD. It was a great oppurtunity to connect with the scientific community and industry partners, and to be updated on the current knowledge of the research field. It was a great learning experience. The PI stayed connected with the industry partners and stakeholders regularly updating them about the project and learning from them about the grower and industry needs. How have the results been disseminated to communities of interest?During the first year of the project, results from the projects were disseminated to scientific community and other stakeholders through annual Pulses meeting organized by USA Pulses. What do you plan to do during the next reporting period to accomplish the goals?Objective#1: 1. Validation of controlled condition screening results for ascochyta blight resistance. Screening of additional wild germplasm for ascochyta blight resistance. Publication of the controlled condition screening results in a peer-review journal. Objective#2: 1. Validation of the Virus-Induced Gene Silencing results in chickpea. 2. Gene Silencing of C-Ph1 gene using TRV-based VIGS vectors. 3. RNA isolation and gene expression analysis to validated virus-induced gene silencing results. Objective#3: 1. Develop and complete segregating germplasm development for genetic mapping of ascochyta blight resistance. 2. Genotype-based sequencing of the mapping population. Objective#4: 1. Field screening of the wild germplasm in replicated trials across Montana, Idaho, and Washington.
Impacts
What was accomplished under these goals?
Objective#1: Identify and confirm novel sources of Ascochyta blightresistance among the wild species: Two independent methods of controlled condition screening were optimized at Geneshifters for screening ascochyta blight resistance. Out of the two methods, initial screening of the 110 wild chickpea accessions was done using the foliar spray method. Additionally, all 110 accessions of chickpea germplasm were being screened by Dr. Weidong Chen using their previously published method. Both approaches have helped in the identification and confirmation of previously identified wild accessions of chickpea for improved resistance compared to currently known sources of resistance. Objective#2: Develop and optimize a novel alien gene transfer approach in chickpea: The optimization of the alien gene transfer method is reliant on the availability of an efficient virus-induced gene silencing system in chickpeas. In order to optimize the virus-induced gene silencing in chickpeas, we are using the Tobacco-Rattle Virus (TRV) system with the PDS gene as a visual marker. 200 bp oligo from the CaPDS gene was cloned to the TRV2-MCS vector prepared after restriction digestion. TRV1, TRV2-MCS, and TRV2-PDS were cloned in the Agrobacterium strain (GV 3101) with appropriate antibiotic selection. GV3101 Agrobacterium strains containing a target plasmid were grown in liquid culture to reach an OD600=1.0. Before starting the agrobacterium culture, about 20 seeds of Swayer were surface sterilized and imbibed for germination. The seeds were allowed to grow till cotyledons emerged. Agrobacterium culture growth and chickpea germination were coordinated to ensure reliable agro-infiltration. The Agrobacterium culture was stopped once it reached desired optical density, and it was resuspended in 10 mM MgCl2 + 10 mM MES + 200 µm Acetosyringone and left at room temperature for 4 hours. TRV is a bipartite virus with two genomic components. Two genomic components were mixed to develop two inoculation cocktails (TRV1 + TRV2-MCS, TRV1 + TRV2-PDS). Using a 1 mL syringe barrel (no needle), the inoculum was delivered to the abaxial side of the cotyledon and infiltrated until the entire cotyledon appeared saturated. The symptoms of photobleaching were observed after 21 days. Objective#3: Develop breeder-friendly markers for the alien gene(s) controlling Ascochyta blightresistance: Mapping population are being developed between Swayer and ascochyta blight resistance donors. About 10 F1s were developed between the Swayer and Cicer pinnatifidum. Objective#4: Field trials to confirm Ascochyta blightresistance and other agronomic traits among develop germplasm: 110 accessions of wild chickpea germplasm were screened for ascochyta blight under field conditions in Bozeman, Montana. 110 accessions of wild chickpea germplasm were screened for ascochyta blight under field conditions in Bozeman, Montana. 110 accessions of wild chickpea germplasm were used for seed multiplication and disease screening at Pullman, Washington. Due to low seed availability, the germplasm was planted in nonreplicated rows of 10ft with spreader rows (Billy beans and Spanish White). All 110 accessions of the germplasm were hand harvested and will be screened in the next planting season in replicated trails for ascochyta blight resistance across three locations.
Publications
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