Source: UNIV OF MARYLAND submitted to
DISSECTION OF ALIEN CHROMOSOMES TO GENERATE USEFUL TRANSLOCATIONS USING GAMMA IRRADIATION INDUCED EXOTIC LIBRARIES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
EXTENDED
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1022771
Grant No.
2020-67013-31460
Project No.
MD-PSLA-05469
Proposal No.
2019-05469
Multistate No.
(N/A)
Program Code
A1141
Project Start Date
Jun 1, 2020
Project End Date
May 31, 2024
Grant Year
2020
Project Director
Tiwari, V. K.
Recipient Organization
UNIV OF MARYLAND
(N/A)
COLLEGE PARK,MD 20742
Performing Department
Plant Science & Landscape Arch
Non Technical Summary
Global population is going to increase to more than nine billion by 2050. To keep pace with the growing demands in wheat production, which are hindered by biotic and abiotic stresses, we will require quick integration of new genes and alleles in the breeding programs. Wild and related germplasm of bread wheat is considered a goldmine of useful genes for wheat improvement, however, precise gene transfer without linkage drag (the integration of undesirable or deleterious genes that negatively influence end-use quality and grain yield in addition to beneficial genes) from wild and related species in wheat is hampered due to lack of genetic recombination, evolutionary chromosomal rearrangements, and lack of genomic resources to detect fine translocations.Here we propose to integrate radiation induced frequent chromosomal breaks with genomic and cytological tools to develop a robust approach for channelizing useful genes from the rich wheat gene pools to bread wheat cultivars. Radiation Hybrid mapping offers a quick approach to create dense maps of the entire alien chromosomes. The exotic libraries containing overlapping alien fragments indexed with diagnostic molecular markers, known fragment size and position of translocations will represent a resource of useful genetic variation recovered from related grasses which are unavailable in cultivated wheat varieties. This will serve as an excellent platform for breeders to incorporate natural variations in the breeding programs with reduced or no linkage drags.
Animal Health Component
0%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20115491081100%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1549 - Wheat, general/other;

Field Of Science
1081 - Breeding;
Goals / Objectives
The long-term goal of this project is to reduce the bottlenecks for the precise gene transfer and develop a pipeline for efficient mapping and transfer of agronomically important genes from distant wild relatives to elite cultivars without linkage drag. The gamma irradiation treatment of pollen samples induces frequent chromosomal deletion and translocation events and can be used to develop recombination independent terminal and interstitial translocation events. This approach will produce high-resolution physical maps of the agronomically important genes or loci and provide linked marker information to track the alien genes in the breeding programs. Radiation Hybrid (RH) mapping offers an efficient path to create dense maps of entire alien chromosomes. Since radiation induced translocations are frequent, by increasing the number of events, multiple interstitial translocation events can be generated. Once a useful translocation is identified, marker assisted recurrent backcrossing with recurrent elite cultivar can be used to remove background chromosomal deletion and other breakage events. The selection of germplasm can be done based on the molecular marker datasets and with the selection of smaller size of the introgressed fragments the penalties of linkage drag will be reduced.ObjectivesDevelopment and initial characterization of exotic- Radiation Hybrid panelsMolecular and cytological characterization of Radiation Hybrid panels and construction of RH mapsIntegration of RH, genomic and cytogenetic data to identify translocationsMarker assisted recurrent backcrossing of selected translocation lines
Project Methods
Objective 1: Development of Exotic-RH PanelsFrom our previous experience with RH panel development, we developed a system of constant planting of donor and recipient RH parental lines on a 2-week rotation. This ensures a constant supply of both male and female plants at the proper developmental stage. All the gamma irradiation experiments will be performed using a Gamma Cell 220 irradiator, at the University of Maryland, College Park. Pollen from the irradiated spikes will be immediately used to pollinate previously emasculated spikes of the recipient lines. For developing more lines (if required) we will use our optimized irradiation dose for development of RH lines for the targeted introgression lines. To develop individual alien chromosome-specific panel, we estimate that we will need to irradiate and pollinate 75-80 spikes with ~15 florets per spike (about 1200- seeds and about 25-30% will germinate to give rise 300-350 plants). The seed from these crosses are termed the RH1 generation and each individual seed represents an independent RH event. After seed is harvested, it will be dried at 37°C for two weeks to remove any latent dormancy and these will be planted in germination boxes for their optimal germination and root tip harvesting.Objective 2: Molecular and Cytological Characterization of RH Panels and Construction of RH MapsTo provide a robust analysis of the RH events, we propose to perform intensive cytological analysis of 300 plants from each RH panel (in total 600 plants). It is important to note that each viable seed generated by crossing irradiated pollens of alien source with wheat recipient female plants represents a unique and independent RH event, so to have the entire panel characterized will provide us statistical power to stipulate the number of total translocation events for a given chromosome in a large pool of samples. After 6-8 days of germination, root tips from the individual RH1 plants will be harvested and will be given same number as the plant from which they are harvested to connect the data from phenotyping, cytology, and genotyping. Two weeks following transplanting of RH1 seedlings, leaf tissue will be collected, and DNA will be extracted per established lab protocols65. Root tips will be collected and placed in cold (1oC) water for 24 hours to disrupt microtubule formation, condense the chromosomes, and increase mitotic index. The root tips will then be squashed and observed under a phase contrast microscope for an initial chromosome count. Slides with a high number of metaphase cells will then be used for GISH. We have identified a small number of alien chromosome specific markers which will be useful in assaying the quality of RH panels and help us in selection of most informative RH lines. Any effort to develop maps translocation segments will depend on identification of new markers. Our latest whole genome RH mapping work clearly indicated that iSelect 90 K SNP genotyping array has a high density of SNP markers and their distribution throughout a given chromosome is robust. Along with SNP based KSAP markers that we have developed from 90K genotyping data, we will use skim sequencing approach to genotype our RH lines. Following the genotyping of RH lines, on iSelect 90K SNP genotyping array, we will construct RH maps of the targeted alien chromosomes. After filtering and genotyping calls, high-quality marker data will be used for grouping and construction of RH maps. Grouping and map construction will be done using the software package Carthagene. Our results on mapping resolutions of RH panel has clearly indicated that map resolution of up to 300Kb is possible using ~300 RH lines. Since our target for each of the translocation line is about 300 RH lines, we think high-resolution mapping of both the phenotype is well within reach and will allow researchers to use this part for molecular isolation of important genes from wheat and other wild crop relatives. Moreover, physical map of the candidate gene regions will allow us to focus on a specific translocation which may carry smallest introgression from alien segment hence reducing linkage drag significantly.Objective 3: Integration of RH, Genomic and Cytogenetic Data to Identify Translocations For mapping purposes, small to large deletions and translocation events are useful, however, for introgression of a specific trait in each cultivar using gamma irradiation, one would expect a mixture of deletion and translocation events for a given chromosome. Gamma irradiation induces libraries of small to large deletions including interstitial and terminal, which will be useful for mapping the resistance of our targeted traits to a chromosomal region, though these events will not be very useful in fine transfer of the resistance in elite cultivars with reduced linkage drag. A large percentage of the chromosomal breakage using gamma irradiation also result in the generation of random translocation events. These translocations can be small or large. In our opinion, most of the useful translocation events in gamma irradiation and Ph1 events are generally lost due to low resolution of the selection approach. Where 1-3 markers per arm in usually tested on a large pool of samples and cytogenetic analysis will be performed on those lines. If that marker is not extremely terminal, then it will not detect a useful translocation, which would be small. Similarly, some useful interstitial translocations will be lost due to inability of marker system to cover entire arm or chromosome. Since we are working on the development of a pipeline, we will start with having an insitu hybridization (GISH) based profile of all RH1 plants. It will allow us to have very clear idea about what to expect in the subsequent generation of the backcrosses that will be made to remove background deletions.Objective 4: Marker Assisted Recurrent Backcrossing of the Selected Translocation Lines Our data cytological characterization of about 600 RH lines from both the panels, molecular marker and its graphical genotype and phenotypic data from RH1 plant as well as from their advanced progenies will provide us robust information to select about 10 small translocation lines from both the panels. Recurrent backcrossing of these lines with recurrent susceptible / elite cultivars will be done, and two flanking markers selected from the high-resolution RH map will be used to perform marker assisted recurrent backcrossing. Our graphical genotyping data will provide us flanking marker information of both the genes and sequences of these flanking markers will be used to develop KASP genotyping assays. These markers will be used to select the targeted introgressions after progeny testing and recurrent back crosses-based background recovery experiments. We will perform screening of 1,128 lines (188 lines x 6 seeds) with multiple markers by PCR. This would require DNA extractions from all 1,128 lines, but with our high throughput DNA extraction robot, we routinely process 400 samples per day and can accomplish this task within two months' time from planting to finish DNA. Based on our preliminary data, we know the arm location of both the traits in this study. The markers will allow identification or tracking of specific translocation in subsequent generations with a minimum of genotyping. We have two large growth chambers assigned to our wheat breeding project. We will use these during the summer- time to speed up marker assisted recurrent backcrossing cycles. After BC4 stage selected lines will be allowed to self and will be used for field testing.

Progress 06/01/22 to 05/31/23

Outputs
Target Audience:Crop breeding, genetic, and genomics community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two postdoctoral scientists, two graduate students and 3 undergraduate students worked on this project. One of the undergraduate students then decided to come back to us as a student in my lab. How have the results been disseminated to communities of interest?Yes, it was achieved through peer-reviewed research publications and invited talks. Tiwari VK: Exploring resistance in winter barley through two independent EMS-induced mutant populations. In: Invited talk at GDER-PBG Virtual conference under Wheat Barley Scab Initiative. (April 27th, 2023) Tiwari VK: Feeding the 10 billion. In: Invited talk at Plant Science and Landscape Architecture Seminar series, University of Maryland, College Park. (April 10th, 2023) Tiwari VK: Plant Breeding Partnership to improve bread wheat. In: Invited talk at Plant and Animal Genome Conference San Diego California USA. (January 14th, 2023) Tiwari VK: Einkorn Genomics for gene discovery and Validation. In: Invited talk at Crop and Soil Science Society of America meeting, Baltimore. (September 6, 2022) Tiwari VK: Combining germplasm, genetics, and genomics tools to enhance FHB resistance in wheat. In: invited talk at the US Wheat Barley Scab Initiative Meeting, Tampa, Florida. (December 5, 2022) Tiwari VK: Speeding up gene discovery in wheat by integrating genomes and germplasm. In: Invited talk at the School of Computational and Integrative Sciences, Jawahar Lal Nehru University, New Delhi, India. (August 1, 2022) Tiwari VK: Integrating forward and reverse genetics tools to develop next-generation wheat cultivars. In: Invited talk at the School of Agricultural Biotechnology, Punjab Agricultural University, Ludhiana, India. (August 2, 2022) Tiwari VK: Combining Genetics and Genomic Tools for Translational Research in Wheat. In: Invited talk at the National Agricultural Biotechnology Institute Mohali, India. (August 4, 2022) Tiwari VK: Progress report on evaluation and development of wheat, barley and triticale cultiavrs for the MD growers and stakeholders. MGPUB Meeting. January 6, 2024 Tiwari VK: Genomics assisted breeding to develop resilient triticale cultivars. In: Inviated talk at Northern Star Seed Meeting, Tampa Florida, December 13, 2023 Tiwari VK: Progress on developing soft red winter wheat cultivars for MD growers. In: Invited talk in MCIA Board meeting. Wye, MD December 12, 2023. Tiwari VK: Development of small grain cultivars for Maryland. In: Inviated talk arranged by Univertisty of Maryland Extension at Cockesville, MD. December 6, 2023 Tiwari VK: Developing climate-resilient triticale cultivars. In: Talk at the Harry Hughes Center of Agroecology grant committee meeting (virtual). (January 23, 2023) Tiwari VK: Progress on 'Developing winter barley cultivars for Maryland'. In: Maryland Grain Producer Board Meeting (January 5, 2023). Tiwari VK: Progress on 'Developing triticale as a cover crop for Maryland'. In: Maryland Grain Producer Board Meeting (January 5, 2023). Tiwari VK: Progress on 'Evaluation of yield and agronomic traits of small grains in Maryland'. In Maryland Grain Producer Board Meeting (January 5, 2023). Tiwari VK: Progress on 'Developing soft red winter wheat cultivars for Maryland'. In: Maryland Grain Producer Board Meeting (January 5, 2023). What do you plan to do during the next reporting period to accomplish the goals?We are working on increasing the seeds of selected translocation lines, and we will then perform field-based testing of developed translocations against diseases.

Impacts
What was accomplished under these goals? 1. Development and initial characterization of exotic- Radiation Hybrid panels To increase the impact of this approach to reduce the alien chromatin and induce translocations in wheat, we developed exotic RH panels of 4 different germplasms.Three different RH panels are being developed for the present study. 1.1 Wheat-rye translocation line 1RS.1BL: The first panel was developed to compare the seed and pollen irradiation treatments for the popular rye 1RS1BL translocation. For this purpose, in 2021, the seeds of the lines created from a cross between wild-type Shirley containing the popular 1RS1BL translocation with the Chinese spring were subjected to a 1.5 Krad dose of gamma radiation. Similarly, a second RH panel was developed using the pollen irradiation method. For this purpose, spikes for the wheat genotype Shirley wild type containing 1RS1BL translocation were irradiated using a 1.5 Krad dose of gamma radiation. 1.2 Wheat-E. trachycaulus translocation line T7AL.7AS-1StS with resistance against Barley Yellow Dwarf Virus (BYDV): Based on our experiment for comparison of seed and pollen irradiation panel, we observed that the pollen panel shows stable translocations and therefore, pollen irradiation method is being used for development and characterization of BYDV radiation panel. A BYDV panel consisting of 400 RH lines were generated and 152 RH1 lines were characterized. For this purpose, wheat-E. trachycaulus translocation line T7AL.7AS-1StS with resistance against Barley Yellow Dwarf Virus (BYDV) was used as a irradiated pollen source. Initially in 2022, about 80 spikes were irradiated using 1.5 Krad Gamma radiation dose; however, most plants were found to be sterile. Therefore, a subsequent experiment was planned with a radiation dose of 1 Krad which provided us with about 600 seeds out of which 400 seeds showed germination and the remaining failed to germinate.These 152 lines are being grown under greenhouse conditions and the leaf tissue has been collected for skim sequencing and molecular marker analysis. The 152 lines will also be subjected to skim sequencing along with the parental BYDV line to identify the potential introgression (Adhikari et al. 2022). Additionally, more lines for BYDV and WL711 have been planted to develop more RH1 lines. 1.3 Wheat-Aegilops ventricosa translocation 2NS/2AS in elite cultivar Jagger:We developed a large population of Jagger cultivars to map valuable genes in the 2NS/2AS region. 900 RH lines were advanced under speed breeding for stable deletion and translocation lines. A large gamma irradiation-based mutant population of Jagger will be used to create overlapping deletion/ translocation libraries of the 2NS segment in the Jagger background. Since the Jagger genome is fully sequenced, we will be detecting fine-scale translocations and deletion events by employing the skim sequencing approach. These tiled-up deletion and translocation events will allow us to fine-map unique disease-resistance genes from 2NS introgressions. A panel of 800 lines is developed and advanced using speed breeding, setting up the R4 generation using single seed descent. After M5 (R5) generation, we will collect the leaf tissue samples from individual M5 plants, and DNA will be extracted to characterize the deletions and translocations using next-generation sequencing. 2. Molecular and cytological characterization of Radiation Hybrid panels and construction of RH maps Wheat-rye translocation line 1RS.1BL.We generated a large deletion population of this translocation line using an elite cultivar, Shirley, that carries the famous 1RS.1BL. A total of 600 lines were generated, and a set of these lines were used for the initial characterization of this panel. We have generated seed as well as pollen irradiation panels. The GISH was performed to identify the rye chromatin in the RH wheat-rye 1BL×1RS line. Fifty-six plants were screened out of which translocation event was presented in 8 plants (Fig. 1), corresponding to % translocation frequency of 14.3%. Dr. Sathishraj Rajendran is currently working on the cytological analysis of each RH-induced plant. The RH-induced plants with shorter rye chromatin fragments will be transferred to agronomically important bread wheat cultivars. These are fascinating results; our translocation frequency is about 15% using a low-resolution GISH approach. It is essential to remember that several studies using chromosome engineering approaches using homoeologous pairing have failed to produce sufficient recombination in this region to transfer valuable genes from rye. Now, we will genotype our RH lines using the skim-sequencing approach to detect a wide range of translocation segments. We are focused on the genotypic and cytological characterization of the more extensive 1RS.1BL translocation lines.For reporting year 3 of the grant, we have achieved all the goals of developing the RH panels and completed the initial characterization. A robust protocol for developing alien chromosome-specific translocation is established. In project years 2- 3, we will focus on mapping and fine-scale characterization of these panels. The following reporting cycle will report Progress on these and other RH lines. 3. Using a low-cost sequencing approach to identify amphidiploid, addition, substitution, and translocation lines: In our project, we have proposed using hybridization-based marker genotyping platforms and exome capture and sequencing as a backup plan.We have demonstrated the success of this approach by testing it over a large set of triticale collections that had a good mixture of octoploid and hexaploid triticale accessions as well as addition or substitution of the rye chromosome in wheat (fig.3). These are fascinating results as this pipeline allow us to detect alien chromosome introgressions using very low-coverage and cheap genotyping by sequencing (GBS) data. Several researchers and geneticists will use this pipeline to establish and identify introgression/translocation events in small grain crop plants. 4. A new approach for mapping alien genes for their precise transfer in wheat: In addition to our work on RH-based translocations, this funding helped optimize and establish a valuable approach for recombination-based reduction of alien segments using homology of the alien segments. As we know, the effective utilization of distant wild relatives in isolation of agronomically essential genes is hindered by the lack of recombination between the homoeologous chromosomes. In this study, we propose a simple yet powerful approach that can be applied for high-resolution mapping of a targeted gene from wheat's distant gene pool members. A wheat-Aegilops geniculata translocation line TA5602 with a small terminal segment from chromosome 5 Mg of Ae. geniculata translocated to 5D of wheat contains genes Lr57 and Yr40 for leaf rust and stripe rust resistance, respectively. To map these genes, TA5602 was crossed with a susceptible Ae. geniculata 5 Mg addition line. Chromosome pairing between the 5 Mg chromosomes of susceptible and resistant parents resulted in developing a high-resolution mapping panel for the targeted genes. Next-generation sequencing data from flow-sorted 5 Mg chromosome of Ae. geniculata allowed us to generate 5 Mg-specific markers. These markers delineated Lr57 and Yr40 genes, each to distinct ~ 1.5 Mb physical intervals flanked by gene markers on 5 Mg. The method presented here will allow researchers worldwide to utilize existing germplasm resources in genebanks and seed repositories toward routinely performing map-based cloning of essential genes from tertiary gene pools of wheat.

Publications

  • Type: Book Chapters Status: Awaiting Publication Year Published: 2024 Citation: Saripalli G, Hosseinirad S, Yadav IS, Kajla A, Schoen AW, Tiwari VK. 2023. Allele mining for improvement of plant architecture, grain quality, and abiotic stress tolerance traits in wheat. In: Allele Mining for Genomic Designing of Cereal Crops. CRC Press. Taylor & Francis (In production)
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Hussain, B.; Akp?nar, B. A.; Alaux, M.; Algharib, A. M.; Sehgal, D.; Ali, Z.; Aradottir, G. I.; Batley, J.; Bellec, A.; Bentley, A. R.; Cagirici, H. B.; Cattivelli, L.; Choulet, F.; Cockram, J.; Desiderio, F.; Devaux, P.; Dogramaci, M.; Dorado, G.; Dreisigacker, S.; Edwards, D.; El-Hassouni, K.; Eversole, K.; Fahima, T.; Figueroa, M.; G�lvez, S.; Gill, K. S.; Govta, L.; Gul, A.; Hensel, G.; Hernandez, P.; Crespo-Herrera, L. A.; Ibrahim, A.; Kilian, B.; Korzun, V.; Krugman, T.; Li, Y.; Liu, S.; Mahmoud, A. F.; Morgounov, A.; Muslu, T.; Naseer, F.; Ordon, F.; Paux, E.; Perovic, D.; Reddy, G. V. P.; Reif, J. C.; Reynolds, M.; Roychowdhury, R.; Rudd, J.; Sen, T. Z.; Sukumaran, S.; Ozdemir, B. S.; Tiwari, V. K.; Ullah, N.; Unver, T.; Yazar, S.; Appels, R.; Budak, H. Capturing Wheat Phenotypes at the Genome Level. Frontiers in Plant Sciences 2022, 13, 851079. https://doi.org/10.3389/fpls.2022.851079.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Yadav, I. S.; Singh, N.; Wu, S.; Raupp, J.; Wilson, D. L.; Rawat, N.; Gill, B. S.; Poland, J.; Tiwari, V. K. Exploring Genetic Diversity of Wild and Related Tetraploid Wheat Species Triticum turgidum and Triticum timopheevii. Journal of Advanced Research 2022. https://doi.org/10.1016/j.jare.2022.08.020.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Arora, S.; Steed, A.; Goddard, R.; Gaurav, K.; OHara, T.; Schoen, A.; Rawat, N.; Elkot, A. F.; Chinoy, C.; Nicholson, M. H.; Asuke, S.; Steuernagel, B.; Yu, G.; Awal, R.; Forner-Mart�nez, M.; Wingen, L.; Baggs, E.; Clarke, J.; Krasileva, K. V.; Tosa, Y.; Jones, J. D. G.; Tiwari, V. K.; Wulff, B. B. H.; Nicholson, P. A Wheat Kinase and Immune Receptor Form the Host-Specificity Barrier against the Blast Fungus. Nature Plants 2023, 9, 385392
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Schoen, A.; Wallace, S.; Meghan, F. H.; Brown-Guidera, G.; Harrison, S.; Murphy, P.; Sanantonio, N.; Van, S. D.; Boyles, R.; Mergoum, M.; Rawat, N.; and Tiwari, V.K. Reducing The Generation Time in Winter Wheat Cultivars Using Speed Breeding. Crop Science.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Schoen, A.; Yadav, I.S.; Poland, J.; Rawat, N.; Tiwari V.K. Genomics Assisted Identification of a Novel Tillering Inhibition Locus tin6 In Hexaploid Wheat. Functional and Integrated Genomics 2023, 12; 23 (2):157
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Kajla, A.; Schoen, A.; Paulson, C.; Kumari, N.; Gill, B.S.; Datla, R.; Coleman, G.; Riera-Lizarazu, O.; Leonard, J.; Rawat, N.; and Tiwari V.K*. High-Resolution Mapping of the C-Locus In Wheat By Combining Radiation Hybrid (RH) And Recombination-Based Mapping Approaches. Theoretical and applied genetics 2023, 136, 159.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Yadav, I.S.; Rawat, N.; Chhuneja, P.; Kaur, S.; Uauy, C.; Lazo, G.; Gu, Y.Q.; Dole~el, J.; and Tiwari, V.K. Comparative Genomic Analysis Of 5Mg Chromosome of Aegilops geniculata and 5Uu chromosome of Aegilops umbellulata Reveal Genic Diversity In The Tertiary Gene Pool. Frontiers in Plant Science. 2023 14 doi: 10.3389/fpls.2023.1144000
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Boyles, RE.: Brown-Guedira, Gina.; Costa.; Cowger, C.; DeWitt, N.; Griffey, C.; Harrison, S.A.; Ibrahim, A.; Johnson, J.; Lyerly, J.; Marshall, D.S.; Mason, E.R; Mergoum, M.; Murphy, J.P.; Winn, Z.; Santantonio, N.; Sutton, R.; Sarripalli, G^.; Tiwari, V.K.; van Sanford, D. 25 Years of Progress Toward Fusarium Head Blight Resistance In Soft Red Winter Wheat. Plant Breeding
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Saripalli, G.; Adhikari, L.; Amos, C.; Kibriya, A.; Ahmed, H.A.; Heuberger, M.; Raupp, J.; Athiyannan, N.K.; Wicker, T.; Abrouk, M.; Wallace, S.; Hosseinirad, S.; Chhuneja, P.; Livesay, J.; Rawat, N.; Krattinger, S.G.; Poland, J.; and Tiwari, V.K. Integration of Genetic and Genomics Resources In Einkorn Wheat Enables Precision Mapping of Key Domestication And Agronomic Traits. Nature Communication Biology
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Ahmed, H.A.; Heuberger, M.; Schoen, A.; Koo, Dal-Hoe.; Quiroz-Chavez, J.; Adhikari, L.; Sharma, P.K.; Raupp, J.; Cauet, S.; Nathalie Rodde, N.; Cravero, C.; Callot, C.; Lazo G.R.; Kathiresan, N.; Athiyannan, N.K.; Yadav I.S ., Moot, I!., Saripalli, G^.; Ramirez-Gonzalez, R.H.; Uauy, C.; Wicker, T.; Abrouk, M.; Tiwari V.K.; Poland, J.; Krattinger, S.G*. Einkorn Genomics Sheds Light on the Evolutionary History of The Oldest Domesticated Wheat. Nature
  • Type: Book Chapters Status: Published Year Published: 2023 Citation: Schoen AW, Saripalli G, Hosseinirad S, Yadav IS, Kajla A, Tiwari VK. 2023. Genome sequences from diploids and wild relatives of wheat for comparative genomics and alien introgressions. In: Genome book. Springer Nature.


Progress 06/01/21 to 05/31/22

Outputs
Target Audience:Crop breeding, genetic, and genomics community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?1. Undergraduate training 2. Graduate student training 3. Delivered invited talks to academic institutions on the project goals and outcomes that were attended by undergraduate, graduate, and postdoctoral scientists. How have the results been disseminated to communities of interest?Yes, it was achieved through peer-reviewed research publications and invited talks. Speeding up gene discovery in wheat by integrating genomes and germplasm. In: Invited talk at the School of Computational and Integrative Sciences, Jawahar Lal Nehru University, New Delhi, India. (August 1, 2022) Integrating forward and reverse genetics tools to develop next-generation wheat cultivars. In: Invited talk at the School of Agricultural Biotechnology, Punjab Agricultural University, Ludhiana, India. (August 2, 2022) Combining Genetics and Genomic Tools for Translational Research in Wheat. In: Invited talk at the National Agricultural Biotechnology Institute (NABI) Mohali, India (August 4, 2022) Integrating genetics and genomics tools to improve wheat for the global grand challenges. In: Invited talk at the department of Biotechnology, Indian Institute of Technology Roorkee, India. (April 25, 2022) Simplifying gene discovery in wheat using diploid forward and reverse genetics Resources. In: International seminar on "Biotechnological Interventions to Overcome the Challenges of Covid/Post-Covid Era (BIOCoPE-2022)" at Amity University Rajasthan, Jaipur, India. (24-25 March 2022) Genomics assisted breeding for improving soft red winter wheat cultivars. In: 2022 SWQL-ARR/SWQC/C&G virtual meeting. USDA-ARS-CSWQRU, Wooster, Ohio. March 15th, 2022. Forward genomics tools for improving yield-related traits in wheat. In Crop Science Seminar Series at the Oregon State University, Corvallis. Feb14th, 2022. Combining forward and reverse genetics tools to identify yield-related genes in wheat. In: Plant and Animal Genome Meeting, San Diego (January 8, 2022) online presentation. Tackling the important issues in small grain cultivars for Maryland and Mid-Atlantic region. In: Commodity Classic, MD (July 26, 2021) Integrating genetics and genomics tools to improve wheat for the grand challenges. In: International Virtual Seminar, Sharda University- Delhi (January 2021). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1. Development and initial characterization of exotic- Radiation Hybrid panels To increase the impact of this approach to reduce the alien chromatin and induce translocations in wheat we developed exotic RH panels of 4 different germplasms. Wheat-rye translocation line 1RS.1BL. Using an elite cultivar Shirley that carries the famous 1RS.1BL, we generated a large deletion population of this translocation line. A total of 600 lines were generated and a set of these lines were used for the initial characterization of this panel. We have generated seed as well as pollen irradiation panels. a): Seed panel: Our initial studies clearly indicated that seed panels are not favored from large-scale translocation events. We found some translocation events in the M1 plants but those were not stable and got filtered out. b): Pollen plant irradiation panel: However, our results from pollen irradiation experiments are very promising. By performing the in-situ hybridization studies of just 40 plants we identified 8 stable and transmissible translocation events. Wheat-Aegilops ventricosa translocation 2NS/2AS in elite cultivar Jagger: To map useful genes in the 2NS/2AS region, we developed a large population of Jagger cultivars. 900 RH lines were advanced under a speed breeding approach to get stable deletion and translocation lines. The large gamma-irradiation-based mutant population of Jagger creates overlapping deletion/ translocation libraries of the 2NS segment in the Jagger background. Since the Jagger genome is fully sequenced, by employing the skim sequencing approach we will be detecting fine-scale translocations and deletion events. These tiled-up deletion and translocation events will allow us to fine-map unique disease-resistance genes from 2NS introgressions. A panel of 800 lines is developed and is advanced using speed breeding setting up to the R4 generation using single seed descend. Wheat-E. trachycaulus translocation line T7AL.7AS-1StS with resistance against Barley Yellow Dwarf Virus (BYDV): Initial evaluation of the panel suggested that the seed irradiation panel may not provide us with stable translocation events. So we optimized pollen irradiation dosages for this Wheat-E. trachycaulus translocation line (T7AL.7AS-1StS). The work on the development is in progress and will be achieved in November 2022. Since evaluation and characterization of the panel would require less than a month time, we are on track to complete this objective on time. 2. Molecular and cytological characterization of Radiation Hybrid panels and construction of RH maps To establish a robust and reproducible protocol for inducing alien chromosome-specific translocations we first started optimization of dosage treatments and found that 1.5Krad treatment is the upper limit for all pollen irradiation experiments. Whereas 35Krad is the upper limit for seed irradiation for several of our tested germplasm. Since the fertility of the samples in pollen irradiation was highly reduced, we first explored seed irradiation treatments to establish our protocol. For this purpose, we generated a total of 600 lines for 1RS.1BL translocation using wheat cultivar Shirley which carries this translocation (a full short arm of rye chromosome 1R was translocated on wheat chromosome arm 1BL) were generated and a set of these lines was used for the initial characterization of this panel (Fig. 1a.). To initially characterize the extent of large translocation events we used Genomic in situ hybridization (GISH), which uses total genomic DNA as a probe, is a powerful tool to discriminate the constituent genomes in natural or artificial hybrids. The GISH was performed to identify the rye chromatin in the RH wheat-rye 1BL×1RS line. Fifty-six plants were screened out of which translocation event was presented in 8 plants (Fig. 1), which corresponds to the translocation frequency of 14.3%. Currently, Dr. Sathishraj Rajendran is working on the cytological analysis of each RH-induced plant. The RH-induced plants having shorter fragments of rye chromatin will be transferred to agronomically important bread wheat cultivars. These are very exciting results, using a low-resolution GISH approach our translocation frequency is about 15%. It is important to remind that several studies using chromosome engineering approaches using homoeologous pairing have failed to produce sufficient recombination in this region to transfer useful genes from rye. Now we will move forward to genotype our RH lines using the skim-sequencing approach to detect a wide range of translocation segments. Now we are focused on the genotypic and cytological characterization of the larger set of the 1RS.1BL translocation lines.For reporting year 2 of the grant, we have achieved all the goals of developing the RH panels and completed the initial characterization, as well as a robust protocol for developing alien chromosome-specific translocation, which is established. In project years 2- and 3, we will focus on mapping and fine-scale characterization of these panels. Progress made on these as well as other RH lines will be reported in the next reporting cycle. 3. Using a low-cost sequencing approach to identify amphiploids, addition, substitution, and translocations lines: In our project, we have proposed to use hybridization-based marker genotyping platforms as well as exome capture and sequencing as a backup plan. But both these approaches are costly and time-consuming. Using a low-cost and very low-coverage Genotyping-by-Sequencing approach, we have developed a pipeline to differentiate gene or genome dosage, addition lines, substitution lines, and translocation lines from alien, wild and related species of wheat. We have demonstrated the success of this approach by testing it over a large set of triticale collections that had a good mixture of octoploid, and hexaploid triticale accessions, as well as the addition or substitution of the rye chromosome in wheat. These, are very exciting results as this pipeline allows us to detect alien chromosome introgressions using very low-coverage and cheap genotyping by sequencing (GBS) data. This pipeline will be exploited by several researchers and geneticists to establish and identify introgression/translocation events in small grain crop plants. 4. A new approach for mapping alien genes for their precise transfer in wheat: In addition to our work on RH-based translocations, this funding helped in optimizing and establishing a useful approach for recombination-based reduction of alien segments using homology of the alien segments. As we know that the effective utilization of distant wild relatives in isolation of agronomically important genes is hindered by the lack of recombination between the homoeologous chromosomes. We propose a simple yet powerful approach that can be applied for high-resolution mapping of a targeted gene from wheat's distant gene pool members. A wheat-Aegilops geniculata translocation line TA5602 with a small terminal segment from chromosome 5 Mg of Ae. geniculata translocated to 5D of wheat contains genes Lr57 and Yr40 for leaf rust and stripe rust resistance, respectively. To map these genes, TA5602 was crossed with a susceptible Ae. geniculata 5 Mg addition line. Chromosome pairing between the 5 Mg chromosomes of susceptible and resistant parents resulted in the development of a high-resolution mapping panel for the targeted genes. The method presented here will allow researchers worldwide to utilize existing germplasm resources in genebanks and seed repositories toward routinely performing map-based cloning of important genes from tertiary gene pools of wheat.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Thudi, M., Palakurthi, R., Schnable, J. C., Chitikineni, A., Dreisigacker, S., Mace, E., Srivastava, R. K. et al. (2020). Genomic resources in plant breeding for sustainable agriculture. Journal of plant physiology, 257, 153351.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Lin, G., Chen, H., Tian, B. et al. Cloning of the broadly effective wheat leaf rust resistance gene Lr42 transferred from Aegilops tauschii. Nat Commun 13, 3044 (2022). https://doi.org/10.1038/s41467-022-30784-9
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: 2. Gaurav, K., Arora, S., Silva, P. et al. (2022). Population genomic analysis of Aegilops tauschii identifies targets for bread wheat improvement. Nat Biotechnol 40, 422431. https://doi.org/10.1038/s41587-021-01058-4
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Habtamu Ayalew, Joshua D Anderson, Nick Krom, Yuhong Tang, Twain J Butler, Nidhi Rawat, Vijay Tiwari, Xue-Feng Ma, Genotyping-by-sequencing and genomic selection applications in hexaploid triticale, G3 Genes|Genomes|Genetics, Volume 12, Issue 2, February 2022, jkab413, https://doi.org/10.1093/g3journal/jkab413
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Chhabra, B., Tiwari V.K., Gill, B.S., Dong, Y., Rawat, N. (2021). Discovery of a susceptibility factor for Fusarium head blight on chromosome 7A of wheat. Theoretical and Applied Genetics. DOI: 10.1007/s00122-021-03825-y.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Chhabra, B., Singh, L., Wallace, S., Schoen, A., Dong, Y., Tiwari, V.K., Rawat, N. (2021). Screening of an EMS mutagenized population of a wheat cultivar susceptible to Fusarium head blight identifies resistant variants. Plant Disease. 105(11):3669-3676
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Steadham, J., Schulden, T., Kalia B., Gill, B.S., Bowden, L., Chhuneja, P., Erwin, J., Tiwari, V.K., Rawat, N. (2021). An approach for high-resolution genetic mapping of distant wild relatives of bread wheat. Theoretical and Applied Genetics 134(8):2671-2686
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Mahlandt, A., Rawat, N., Leonard, J., Venglat, P., Datla, R., Meier, N., Gill, B.S., Riera-Lizarazu, O., Coleman, G., Murphy, A.S., Tiwari, V.K. (2021). High-resolution mapping of the Mov-1 Locus in wheat by combining Radiation-Hybrid and recombination-based mapping approaches. Theoretical and Applied Genetics. DOI: 10.1007/s00122-021-03827-w.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Rabanus-Wallace, M.T., Hackauf, B., Mascher, M. et al. Chromosome-scale genome assembly provides insights into rye biology, evolution and agronomic potential. Nat Genet 53, 564573 (2021). https://doi.org/10.1038/s41588-021-00807-0


Progress 06/01/20 to 05/31/21

Outputs
Target Audience:Crop breeding, genetic, and genomics community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?1. Undergraduate training 2. Graduate student training 3. Delivered invited talks to academic institutions on the project goals and outcomes that were attended by undergraduate, graduate, and postdoctoral scientists. How have the results been disseminated to communities of interest?Yes, it was achieved through peer-reviewed research publications and invited talks. Publications Chhabra, B.†, Tiwari V.K., Gill, B.S., Dong, Y., Rawat, N.* (2021). Discovery of a susceptibility factor for Fusarium head blight on chromosome 7A of wheat. Theoretical and Applied Genetics. DOI: 10.1007/s00122-021-03825-y. Chhabra, B.†, Singh, L.†, Wallace, S.†, Schoen, A., Dong, Y., Tiwari, V.K., Rawat, N.* (2021). Screening of an EMS mutagenized population of a wheat cultivar susceptible to Fusarium head blight identifies resistant variants. Plant Disease. Accepted. Steadham, J.†, Schulden, T.†, Kalia B., Gill, B.S., Bowden, L., Chhuneja, P., Erwin, J., Tiwari, V.K., Rawat, N.* (2021). An approach for high-resolution genetic mapping of distant wild relatives of bread wheat. Theoretical and Applied Genetics 134(8):2671-2686Mahlandt, A., Rawat, N., Leonard, J., Venglat, P., Datla, R., Meier, N., Gill, B.S., Riera-Lizarazu, O., Coleman, G., Murphy, A.S., Tiwari, V.K.*. (2021). High-resolution mapping of the Mov-1 Locus in wheat by combining Radiation-Hybrid and recombination-based mapping approaches. Theoretical and Applied Genetics. DOI: 10.1007/s00122-021-03827-w. International Rye Genome Sequencing Consortium. Chromosome-scale genome assembly provides insights into rye biology, evolution, and agronomic potential. Nature Genetics. (In-press) Thudi, M., Palakurthi, R., Schnable, J. C., Chitikineni, A., Dreisigacker, S., Mace, E., Srivastava, R. K. et al. (2020). Genomic resources in plant breeding for sustainable agriculture. Journal of plant physiology, 257, 153351. Published Invited talks: Integrating genetics and genomics tools to improve wheat for the grand challenges. In: International Virtual Seminar, Sharda University- Delhi (January 2021). Wheat Genomes, Transcriptomes, and Beyond. In: Weekly Research. In: Progress Seminar series in the Center for Bioinformatics and Computational Biology at the University of Maryland, College Park (December 2020). Wheat Genetics and Genomics in the post-reference genome era. In: International e-Conference on Genetics and Plant Breeding Research in Post COVID-19 Era (June 2020). CCSU, Meerut, India What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Hiring: Two postdoctoral scientists (one full-time postdoc at UMD and another at K-State with 25% time supported by this grant) have been hired under this grant. Dr. Gautam Saripalli will be joining the lab at the University of Maryland, College Park, by end of this summer. Currently, Dr. Sathishraj Rajendran at the Kansas State University has been supported by this grant to work on the cytological analysis of each of RH-induced plants. Since the postdoctoral scientist was not able to join until September 2021 the project due to Covid-19 related issues, a graduate student was charged to work on it to timely achieve the project objectives. Fortunately, we hired Dr. Sathishraj Rajendran at the K-State, who is funded for 30% of his time working on this grant. He is working on the cytological analysis of each RH-induced plant. Progress made against project goals: 1. Development and initial characterization of exotic- Radiation Hybrid panels To increase the impact of this approach to reduce the alien chromatin and induce translocations in wheat we developed exotic RH panels of 4 different germplasms. Wheat-rye translocation line 1RS.1BL. Using an elite cultivar Shirley that carries the famous 1RS.1BL, we generated a large RH population of this translocation line. A total of 600 lines were generated and a set of these lines were used for the initial characterization of this panel. Wheat-Aegilops ventricosa translocation 2NS/2AS in elite cultivar Jagger: We developed large gamma irradiation-based mutant population of Jagger to create overlapping deletion/ translocation libraries of the 2NS segment in the Jagger background. Since the Jagger genome is fully sequenced, we will be detecting fine-scale translocations and deletion events by employing the skim sequencing approach. These tiled-up deletion and translocation events will allow us to fine map unique diseases resistance genes from 2NS introgressions. A panel of 800 lines is developed and is advanced using speed breeding setting up to the R4 generation. Wheat-E. trachycaulus translocation line T7AL.7AS-1StS with resistance against Barley Yellow Dwarf Virus (BYDV): To reduce the alien chromosome segment and develop compensating translocation of 1StS segment from Elymus trachycaulus we irradiated 1000 seeds using our modified protocol and an RH population of 400 lines was generated. 2. Molecular and cytological characterization of Radiation Hybrid panels and construction of RH maps To establish a robust and reproducible protocol for inducing alien chromosome-specific translocations we first started optimization of dosage treatments and found that 1.5Krad treatment is the upper limit for all pollen irradiation experiments. Whereas 35Krad is the upper limit for seed irradiation for several of our tested germplasm. Since the fertility of the samples in pollen irradiation was highly reduced, we first explored seed irradiation treatments to established our protocol. For this purpose, we generated a total of 600 lines for 1RS.1BL translocation using wheat cultivar Shirley which carries this translocation (a full short arm of rye chromosome 1R was translocated on wheat chromosome arm 1BL) were generated and a set of these lines were used for the initial characterization of this panel. To initially characterize the extent of large translocation events we used Genomic in situ hybridization (GISH), which uses total genomic DNA as a probe, is a powerful tool to discriminate the constituent genomes in natural or artificial hybrids. The GISH was performed to identify the rye chromatin in the RH wheat-rye 1BL×1RS line. Fifty-six plants were screened out of which translocation event was presented in 8 plants, which corresponds to the translocation frequency of 14.3%. Currently, Dr. Sathishraj Rajendran is working on the cytological analysis of each RH-induced plant. The RH-induced plants having shorter fragments of rye chromatin will be transferred to agronomically important bread wheat cultivar. These are very exciting results, using a low-resolution GISH approach our translocation frequency is about 15%. It is important to remind that several studies using chromosome engineering approaches using homoeologous pairing have failed to produce sufficient recombinations in this region to transfer useful genes from rye. Now we are focused on the genotypic and cytological characterization of the larger set of the 1RS.1BL translocation lines.For reporting year 1 of the grant we have achieved all the goals of developing the RH panels and completed the initial characterization as well as a robust protocol for developing alien chromosome-specific translocation is established. In project years 2- and 3, we will focus on mapping and fine-scale characterization of these panels. Progress made on these as well as other RH lines will be reported in the next reporting cycle. 3. A new approach for mapping alien genes for their precise transfer in wheat: In addition to our work on RH-based translocations, this funding helped in optimizing and establishing a useful approach for recombination-based reduction of alien segments using homology of the alien segments. As we know that the effective utilization of distant wild relatives in isolation of agronomically important genes is hindered by the lack of recombination between the homoeologous chromosomes. In this study, we propose a simple yet powerful approach that can be applied for high-resolution mapping of a targeted gene from wheat's distant gene pool members. A wheat-Aegilops geniculata translocation line TA5602 with a small terminal segment from chromosome 5 Mg of Ae. geniculata translocated to 5D of wheat contains genes Lr57 and Yr40 for leaf rust and stripe rust resistance, respectively. To map these genes, TA5602 was crossed with a susceptible Ae. geniculata 5 Mg addition line. Chromosome pairing between the 5 Mg chromosomes of susceptible and resistant parents resulted in the development of a high-resolution mapping panel for the targeted genes. Next-generation-sequencing data from flow-sorted 5 Mg chromosome of Ae. geniculata allowed us to generate 5 Mg-specific markers. These markers were used to delineate Lr57 and Yr40 genes each to distinct ~ 1.5 Mb physical intervals flanked by gene markers on 5 Mg. The method presented here will allow researchers worldwide to utilize existing germplasm resources in genebanks and seed repositories toward routinely performing map-based cloning of important genes from tertiary gene pools of wheat.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: 1. Chhabra, B. , Tiwari V.K., Gill, B.S., Dong, Y., Rawat, N.* (2021). Discovery of a susceptibility factor for Fusarium head blight on chromosome 7A of wheat. Theoretical and Applied Genetics. DOI: 10.1007/s00122-021-03825-y.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: 2. Chhabra, B. , Singh, L. , Wallace, S. , Schoen, A., Dong, Y., Tiwari, V.K., Rawat, N.* (2021). Screening of an EMS mutagenized population of a wheat cultivar susceptible to Fusarium head blight identifies resistant variants. Plant Disease. Accepted.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: 3. Steadham, J. , Schulden, T. , Kalia B., Gill, B.S., Bowden, L., Chhuneja, P., Erwin, J., Tiwari, V.K., Rawat, N.* (2021). An approach for high-resolution genetic mapping of distant wild relatives of bread wheat. Theoretical and Applied Genetics 134(8):2671-2686
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: 5. Mahlandt, A., Rawat, N., Leonard, J., Venglat, P., Datla, R., Meier, N., Gill, B.S., Riera-Lizarazu, O., Coleman, G., Murphy, A.S., Tiwari, V.K.*. (2021). High-resolution mapping of the Mov-1 Locus in wheat by combining Radiation-Hybrid and recombination-based mapping approaches. Theoretical and Applied Genetics. DOI: 10.1007/s00122-021-03827-w.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Rabanus-Wallace, M.T., Hackauf, B., Mascher, M. et al. Chromosome-scale genome assembly provides insights into rye biology, evolution, and agronomic potential. Nat Genet 53, 564573 (2021). https://doi.org/10.1038/s41588-021-00807-0