NEW SEMI-DWARF ALLELES TO IMPROVE YIELD AND QUALITY OF BREAD WHEAT AND DURUM

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1018537
• Grant No.: 2019-67014-29199
• Cumulative Award Amt.: $500,000.00
• Proposal No.: 2018-06173
• Project Start Date: May 1, 2019
• Project End Date: Apr 30, 2023
• Grant Year: 2019
• Program Code: [A1141]- Plant Health and Production and Plant Products: Plant Breeding for Agricultural Production

Recipient Organization
MONTANA STATE UNIVERSITY
(N/A)
BOZEMAN,MT 59717

Performing Department
Plant Sciences and Plant Path.

Non Technical Summary
Plant height is an important characteristic in wheat. Modern semi-dwarf varieties have higher yields compared to standard height wheat varieties, yet there is limited genetic variation for this trait. Essentially all semi-dwarf wheat varieties contain one of two mutant forms of the Reduced Height (Rht) gene. Intermediate height alleles are desirable in hexaploid wheat and durum to maximize yield, protein content, and product quality, yet do not exist in modern germplasm. The goal of this proposal is to overcome the lack of Rht allelic diversity in bread wheat and durum. Lack of wheat Rht allelic diversity is an obstacle to increasing yield and product quality and will continue to remain so in the face of climate change. This project will result in superior wheat phenotypes that will benefit growers nationwide. In addition to optimizing height, new Rht allelic combinations will improve coleoptile length for increased stand establishment and improve yield and grain quality in bread and durum. This project addresses the priority of Plant Breeding for Agricultural Production Program Area Priority Code-A1141.

Animal Health Component

40%

Research Effort Categories

Basic

60%

Applied

40%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
202	1549	1081	100%

Knowledge Area
202 - Plant Genetic Resources;

Subject Of Investigation
1549 - Wheat, general/other;

Field Of Science
1081 - Breeding;

Keywords

adaptation

genetic resource

plant breeding

wheat

Goals / Objectives
Plant height is an important characteristic in wheat. Modern semi-dwarf varieties have higher yields compared to standard height wheat varieties, yet there is limited genetic variation for this trait. Essentially all semi-dwarf wheat varieties contain one of two mutant forms of the Reduced Height (Rht) gene. Intermediate height alleles are desirable in hexaploid wheat and durum to maximize yield, protein content, and product quality, yet do not exist in modern germplasm. The goal of this proposal is to overcome the lack of Rht allelic diversity in bread wheat and durum.Our specific objectives are:1. Integration of Rht-A1, Rht-B1, and Rht-D1 C-terminal stop codon alleles into spring and winter wheat. Year 1 through Year 2. This will result in BC2F2 plant material ready for transfer to breeding programs.2. Yield and product quality testing of BC2F2 lines to identify Rht allelic combinations best suited to optimize wheat yield and quality. Year 2 through Year 3. This will identify ideal allelic combinations for target environments.3. Identify and characterize novel Rht-A1 and Rht-B1 alleles in durum. Years 1 through 3. This includes evaluation of plant yield related characters.Promising alleles will be crossed into select varieties for preparation of transfer into breeding programs.Lack of wheat Rht allelic diversity is an obstacle to increasing yield and product quality and will continue to remain so in the face of climate change. This project will result in superior wheat phenotypes that will benefit growers nationwide. In addition to optimizing height, new Rht allelic combinations will improve coleoptile length for increased stand establishment and improve yield and grain quality in bread and durum.

Project Methods
Objective 1: Year 1 through Year 2. In this objective, we will continue toward integrating unique Rht allelic combinations into elite wheat germplasm. Plant height phenotypes within the zones of interest will be obtained by intercrossing Rht variant alleles alone and in combination with Rht-B1b. We will cross select alleles into spring and winter varieties for preliminary testing and transfer to breeding programs. Development of BC2F2 populations: For each of the variant alleles, M3 plants containing the Rht mutation were crossed to the parent variety Fortuna. The resultant F1 plants were then advanced to the F2 generation for preliminary analysis. These F2 lines were then crossed with each spring and winter variety to advance all genotypes to BC2F2 in spring and winter wheat backgrounds.Progress in spring wheat: We have achieved our target BC2F2 semi-dwarf genotypes for individual Rht alleles of interest in Fortuna and Duclair. In addition to advancing seed to the BC2F2 generation and identification of homozygotes, crossing efforts will focus on creation of the Rht-A-stop/D-stop mutant as well as triple mutants in both backgrounds. In Duclair, crossing is slightly more complicated due to segregation between Rht-Bstop and Rht-B1b. Since Rht-B1b is already present, crossing of Rht-A-stop and Rht-D-stop will result in double allele combinations, therefore the only single mutant allele combination that will be obtained is the Rht-B-stop. Presence of Rht-B1b will also result in additional double and triple allele combinations.Integrating Rht variant alleles into winter wheat: In integrating selected new Rht alleles into winter wheat, we must also select plants that are homozygous for winter growth habit. Spring versus winter growth habit in wheat is governed by three orthologous Vrn-1 loci on homoeologous group 5 chromosomes. Spring habit (no vernalization requirement) can be obtained with a dominant allele at least one of the Vrn-1 loci. We have screened our spring and winter wheat parents to determine which dominant spring allele is carried by the spring parents (generally only dominant Vrn allele is present) and which marker(s) must be used in crossing.Progress in winter wheat: As in the spring wheat Duclair background, the Rht-B1b semi-dwarf allele is already present in the chosen winter backgrounds. Each of the three winter wheat varieties have been crossed to the Rht-Astop (A-319) line and are in the BC1F1 generation. For addition of Rht-A1stop, a BC1F3 Fortuna line was crossed to each of the winter varieties. For addition of both A1stop and D1stop, in spring and winter wheat, we will make use of the BC1F1 populations to cross double mutants into, to reduce background mutation levels.OBJECTIVE 2: Year 2 throughYear 3. Detailed evaluation of agronomic and end product quality traits for the BC2F2 lines produced in Objective 1. Phenotypic analysis will include evaluation of plant growth, yield, and seed development under standard agronomic conditions. This objective will allow us to test the degree to which our novel alleles and allele combinations impacts agronomic yield or seed quality compared to Rht-B1b, Rht-D1b, or standard height wheat. Quality testing will examine the effect of Rht allele presence on milling and baking properties.Dryland vs Irrigated: This experiment will take place under two environments: dryland and irrigated at two growing locations. In the irrigated environment, water will be delivered twice, one week before and after anthesis. The amount delivered will depend on growing location.Statistical Analysis: For yield as well as end product quality traits, each response variable will be analyzed using analysis of variance under a model for a randomized complete block design combined over environments. All factors will be considered fixed and comparisons among genotypic means will be performed using Least Significant Difference (LSD) with the 'Agricolae' R package.OBJECTIVE 3: Year 1 through Year 3. Durum tends to be taller than most bread wheat varieties and is best suited for growth under dryland agronomic conditions. Evidence indicates that current semi-dwarf durum varieties (containing Rht-B1b) are slightly too short, and that maximum tiller number has not been achieved under normal growth conditions. Creation of height alleles resulting in slightly taller semi-dwarf durum will allow for yield increases associated with increased tiller number. Therefore, we are expanding this project to include identification and characterization of Rht-A1and Rht-B1 missense alleles in durum. Our goal is to identify new allelic variants of intermediate height between tall varieties and currently available semi-dwarf varieties (Rht-B1b). This will result in durum better suited for growth under dryland production practices.Population selection for screening of novel Rht-1 alleles in durum: This objective will be accomplished by screening two separate populations. Seed from the standard height durum variety 'Divide' and the semi-dwarf variety 'Alzada' will be mutagenized by soaking seed in a solution of 1% EMS. The tall 'Divide' population contains the wild type Rht-A1a and Rht-B1a alleles, while 'Alzada' contains Rht-B1b. In Alzada, we will only select for the new variant Rht-Bstop allele, since we are not interested in creating double mutant combinations using Rht-B1b (resulting phenotypes would be shorter than desired). The combination of searching for mutants in both directions will ensure that we capture novel alleles for intermediate height and will ensure success of this objective.Screening for mutations at the Rht-1 loci: We will use a combination of two approaches to screen for seed. In the Divide population, we will select for plants that have shorter coleoptiles and have reduced height compared to the average non-mutagenized control. Seed will be germinated in germination paper rolls placed in either water (control) or 10-5 M gibberellic acid and then placed in a darkened growth chamber. In the short Alzada population, we will select for plants with longer coleoptiles and increased height (hypomorphic) compared to the control. Seed will be germinated in the dark since coleoptiles stop extending once light is detected. Coleoptile length will be recorded once maximum length has been reached (~10-15 days after planting, once first leaf has emerged). Plant height will be measured at maturity and plants with coleoptile lengths and plant heights intermediate between Rht-B1b and the WT will be sequenced for mutations in Rht-A1 and Rht-B1 as described in the preliminary results. Relative impact of the identified mutations will be estimated by calculating a Provean score.Advancement of mutant lines: To reduce background mutation levels, M1 plants will be crossed into elite germplasm. We have selected recurrent parents MT 122119 and MTD 16005 based on their yield potential and low cadmium uptake. These lines represent both standard tall and semi-dwarf phenotypes: MT 12119 is a semi-dwarf containing the Rht-B1b allele, while MTD 16005 is tall with the Rht-B1a allele. In addition, we will intercross variant alleles in the same manner as for bread wheat (outlined under Objective 1). All genotypes of interest will be advanced to the BC2F2 generation. Each round of crossing is expected to be completed within a 4 month time frame.Evaluation of plant yield related characters: Initial characterization of mutant lines will occur during each round of crossing. Measurements that will be collected include plant height, tiller number, leaf length and width, seed weight per plant, and individual seed weight. Since we are not interested in plants that are shorter than current semi-dwarf plants, we are not interested in double allele combinations containing Rht-B1b. Preliminary field testing of plant growth and yield will during the third year of funding on BC2F2 plants.

Progress 05/01/19 to 04/30/23

Outputs
Target Audience:The target audience for this project is plant scientists interested in genes that impact plant growth and development andincludes plant breeders and geneticists. The information gained in working toward the project's goal of increasing yield in wheat via a better understanding of wheat semi-dwarf genes could readily be applied to other agronomic groups. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Four graduate students and fiveundergraduate students have been trained. Each of the graduate students and the undergraduates has been given the opportunity to present their results in lab meetings and group meetings at Montana State. The first Ph.D. student graduated in December 2019, an MS student graduated in fall 2020 and a new MS student started August 2020 who graduated February 2023. an additional MS student started January 2021 who has since become a PhD student and another Ph.D. student started fall 2021. How have the results been disseminated to communities of interest?The results have beenpresented in poster and oral presentations at regional and national meetings. This past spring and summer the results have been presented informally to local wheat grower groups at our in-person field days and in 2022 atthe national plant breeding meeting. What do you plan to do during the next reporting period to accomplish the goals?We are continuing to work on replicated field trials, though we are now beyond the scope of the original proposal and the funding period has ended.

Impacts
What was accomplished under these goals? We completed a study that presents the creation, isolation, and initial characterization of new alleles of Rht-A1, Rht-B1, and Rht-D1. Stop codons in each of the three genes were characterized along with several missense alleles in terms of their impact upon binding to GID1. Binding to GID1 is a way to measure RHT function in vitro. We integrated these new alleles into spring and winter wheat and have completed the initial characterization of a set of new Rht alleles in durum wheat. Initial field tests in durum wheat indicate that height intermediate between standard height and semi-dwarf is possible with new Rht alleles and that would directly lead to increased yield under moderate to low yielding environments. These experiments require several rounds of backcrossing and preliminary field testing and we are progressing toward full density full trials. The first trials were completed in 2022 and we are carrying forward additional trials in 2023. The isolines in several backgrounds are in the field this summer in several locations and indicate that we have three alleles conferring intermediate height in durum wheat. These three alleles should increase seed size and protein content relative to Rht-B1b. Testing will continue in spring and winter wheat.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Vetch, J.M., B.J. Tillett, P. Bruckner, J.M. Martin, K. Marlowe, M.A. Hooker, D.R. See, and M.J. Giroux. (2022). TAMFT-3A and TAMFT-3B2 homeologs are associated with wheat preharvest sprouting. Plant Genome 15:e20250 https://doi.org/10.1002/tpg2.20250
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Brown, M.M., J.M. Martin, E.M. Jobson, A.C. Hogg, P.M. Carr, and M.J. Giroux. (2022). Evaluating the impact of Rht hypomorphic mutations in durum wheat. Crop Science 62, 247-258. https://doi.org/10.1002/csc2.20672
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Martin, J.M., J. Zhang, A.C. Hogg, and M.J. Giroux. (2023). Missense alleles of the HMW Glutenin subunits Dx5 and Dy10 have small changes in function relative to missense changes in Puroindoline a and b. Cereal Chemistry 100:171182. https://doi.org/10.1002/cche.10628

Progress 05/01/21 to 04/30/22

Outputs
Target Audience:The target audience for this project is plant scientists interested in genes that impact plant growth and development and includes plant breeders and geneticists. The information gained in working toward the project's goal of increasing yield in wheat via a better understanding of wheat semi-dwarf genes could readily be applied to other agronomic groups. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three graduate students and twoundergraduate students have been trained. Each of the graduate students and the undergraduates has been given the opportunity to present their results in lab meetings and group meetings at Montana State. The first Ph.D. student graduated in December 2019, an MS student graduated in fall 2020 and a new MS student started August 2020, an additional MS student started January 2021 and a Ph.D. student started fall 2021. There are currently two PhD students (one of the MS students converted to a PhD) and the other PhD student will graduate at the end of 2022. How have the results been disseminated to communities of interest?The results are typically presented in poster and oral presentations at national scientific meetings and that is again finally possible in summer 2022. This past spring and summer the results have been presented informally to local wheat grower groups at our in-personfield days and in will be done in person at the national plant breeding meeting summer 2022. What do you plan to do during the next reporting period to accomplish the goals?The alleles conferring unique plant traits were backcrossed into spring, winter, and durum wheat. Isolines have been made in two different spring and durum varieties for Rht alleles varying in function. The winter isolines are still being completed. The first tests of isolines under field conditions is taking place this summer with full density field trials planned for summer 2023.

Impacts
What was accomplished under these goals? We completed a study that presents the creation, isolation, and initial characterization of new alleles of Rht-A1, Rht-B1, and Rht-D1, and the paper has now been published. Stop codons in each of the three genes were characterized along with several missense alleles in terms of their impact upon binding to GID1. Binding to GID1 is a way to measure RHT function in vitro. We are continuing to integrate these new alleles into spring and winter wheat and have completed the initial characterization of a set of new Rht alleles in durum wheat. Initial field tests in durum wheat indicate that height intermediate between standard height and semi-dwarf is possible with new Rht alleles and that would directly lead to increased yield under moderate to low yielding environments. These experiments require several rounds of backcrossing and preliminary field testing and we are progressing toward full density full trials. The isolines in several backgrounds are in the field this summer and indicate that we have three alleles conferring intermediate height in durum wheat. These three alleles should increase seed size and protein content relative to Rht-B1b.

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2022 Citation: Vetch, J.M., B. Tillett, J.M. Martin, A.C. Hogg, and M.J. Giroux. TaMFT homeologs are associated with preharvest sprouting in winter wheat. Plant Genome, July 2022, accepted.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2022 Citation: Brown, M.M., J.M. Martin, A.C. Hogg, L. Wright, C. Hale, P.M. Carr, and M.J. Giroux. Teosinte Branched 1 mutations increase tillering. Crop Science, accepted June 2022.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2022 Citation: Brown, M.M., J.M. Martin, E.M. Jobson, A.C. Hogg, P.M. Carr, and M.J. Giroux. (2022). Evaluating the impact of Rht hypomorphic mutations in durum wheat. Crop Science 62, 247-258. https://doi.org/10.1002/csc2.20672

Progress 05/01/20 to 04/30/21

Outputs
Target Audience:The target audience for this project are plant scientists interested in genes that impact plant growth and development and includes plant breeders and geneticists. Information gained in working toward the project's goal of increasing yield in wheat via a better understanding of wheat semi-dwarf genes could readily be applied to other agronomic groups. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three graduate students and four undergraduate students have been trained. Each of the graduate students and the undergraduates havebeen given the opportunity to present their results in lab meetings and group meetings at Montana State.The first Ph.D. student graduated in December 2019, an MS student graduated in fall 2020 and a new MS student started August 2020, an additional MS student started January 2021 and a Ph.D. student will start August 2021. How have the results been disseminated to communities of interest?The results are typicallypresented in poster and oral presentations at national scientific meetings but that wasnot possible due to travel restrictions in place since spring 2020. However, the results have been presented informally to local wheat grower groups at our virtual field day and in summer 2020 this was done via virtual field day presentations by both the PI and one of the MS students. Summer 2021 will have in-person field day presentations by myself and the graduate students and virtual presentations at two regional meetings. What do you plan to do during the next reporting period to accomplish the goals?The yeast 2 hybrid studies and F2 populations in spring and durum wheat have helped us to define the Rht alleles that are the most valuable to pursue. The alleles conferring unique plant traits are being backcrossed into spring, winter, and durum varieties and proceeding toward field testing.

Impacts
What was accomplished under these goals? We have completed a study that presents the creation, isolation, and initial characterization of new alleles of Rht-A1, Rht-B1, and Rht-D1 and the paper has now been published. Stop codons in each of the three genes were characterized along with several missense alleles in terms of their impact upon binding to GID1. Binding to GID1 is a way to measure RHT function in vitro. We are continuing to integrate these new alleles into spring and winter wheat and have completed the initial characterization of a set of new Rhtalleles in durum wheat. Initial field tests in durum wheatindicatethat height intermediate between standard height and semi-dwarf is possible with new Rhtalleles and that would directly lead to increased yield under moderate to low yielding environments. These experiments require several rounds of backcrossing and preliminary field testing and we are progressing toward full density full trials.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Jobson, E.M., J.M. Martin, R. Sharrock, A.C. Hogg, and M.J. Giroux. (2021a) Identification and molecular characterization of novel Rht-1 alleles in hard red spring wheat. Crop Science. 2021;61:10301037. https://doi.org/10.1002/csc2.20375
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Jobson, E.M., J-B. Ohm, J.M. Martin, and M.J. Giroux. (2021b) Rht-1 semi-dwarfing alleles increase the abundance of high molecular weight glutenin subunits. Cereal Chemistry 98:337-345.

Progress 05/01/19 to 04/30/20

Outputs
Target Audience:The target audience for this project are plant scientists interested in genes that impact plant growth and development and includes plant breeders and geneticists. Information gained in working toward the project's goal of increasing yield in wheat via a better understanding of wheat semi-dwarf genes could readily be applied to other agricultural crops. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students and four undergraduate students have been trained. Each of the graduate students and the undergraduates have been given the opportunity to present their results at local, regional, or national meetings. The first PhD student graduated December 2019, a MS student is on track to graduate fall 2020 and a new MS student is ready to start August 2020. How have the results been disseminated to communities of interest?The results have been presented in poster and oral presentations at national scientific meetings. The results have also been presented informally to local wheat grower groups. In summer 2020 this was done via virtual field day presentations by both the PI and one of the MS students. What do you plan to do during the next reporting period to accomplish the goals?The yeast 2 hybrid studies and F2 populations in spring and durum wheat have helped us to define the Rht alleles that are the most valuable to pursue. The alleles conferring unique plant traits are being backcrossed into spring, winter, and durum varieties and proceeding toward field testing.

Impacts
What was accomplished under these goals? We have completed a study that presents the creation, isolation, and initial characterization of new alleles of Rht-A1, Rht-B1, and Rht-D1. Stop codons in each of the three genes were characterized along with several missense alleles in terms of their impact upon binding to GID1. Binding to GID1 is a way to measure RHT function in vitro. Excitingly, we found several alleles that result in an intermediate level of binding between RHT and GID1. That is potentially useful in that those alleles would create intermediate height genotypes that would be potentially useful in different environments. We are continuing to integrate these new alleles into spring and winter wheat and have completed the initial characterization of a set of new RHT alleles in durum wheat. Initial field test indicates that height intermediate between standard height and semi-dwarf is possible with these new alleles and that would directly lead to increased yield under moderate to low yielding environments. These experiments requireseveral rounds of backcrossing and preliminary field testing and we expect to progress to full density yield trials in years 2 and 3.

Publications

• Type: Journal Articles Status: Submitted Year Published: 2020 Citation: Jobson, E.M., J.M. Martin, R. Sharrock, A.C. Hogg, and M.J. Giroux. (2020b) Identification and molecular characterization of novel Rht-1 alleles in hard red spring wheat. Crop Science, Submitted February 2020.
• Type: Journal Articles Status: Submitted Year Published: 2020 Citation: Jobson, E.M., J-B. Ohm, J.M. Martin, and M.J. Giroux. (2020a) Rht-1 Semi-dwarfing alleles alter dough rheology by modifying gluten component protein composition. Cereal Chemistry, Submitted February 2020.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Jobson, E.M., R.E. Johnston, A.J. Oiestad, J.M. Martin and M.J. Giroux. 2019. The impact of the wheat Rht-B1b semi-dwarfing allele on photosynthesis and seed development under field conditions. Frontiers in Plant Science 10(51). https://doi.org/10.3389/fpls.2019.00051