PARTNERSHIP: TRITICALE RESEARCH AND BREEDING FOR GRAIN QUALITY AND YIELD

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1030046
• Grant No.: 2023-67013-39297
• Cumulative Award Amt.: $800,000.00
• Proposal No.: 2022-10296
• Project Start Date: Jan 1, 2023
• Project End Date: Dec 31, 2026
• Grant Year: 2023
• Program Code: [A1141]- Plant Health and Production and Plant Products: Plant Breeding for Agricultural Production

Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671

Performing Department
(N/A)

Non Technical Summary
In an era of rapid population growth, increasing fertilizer prices and limited water for agriculture, it is essential to maximize the efficiency of food production per unit input or acre. Triticale yields are on average 11% higher than wheat (20% under nitrogen stress) and triticale varieties are currently grown on over 2 million acres in the US and over 10 million acres globally. However, insufficient breadmaking quality has limited triticale use to forage and feed applications. In this project, we propose to improve triticale breadmaking quality with the ultimate goal of unlocking the use of this highly productive crop for direct human food consumption. We have already developed lines carrying different combinations of five genes from wheat known to have favorable effects on breadmaking quality. These lines are currently being tested by artisan millers and bakers. Our first objective is to evaluate the effect of these introgressed loci on breadmaking quality and grain yield. Our second and third objectives are to identify chromosome region responsible for the exceptional loaf volume previously identified in two Ukrainian triticale lines, validate them, and deploy them in existing breeding populations across the US and CIMMYT. Finally, we propose to combine these new chromosome regions with favorable effects on breadmaking quality with the five known positive wheat quality loci we previously introgressed into triticale lines. This work will be conducted by a collaborative triticale network including California, Washington, Colorado, Nebraska, Maryland and CIMMYT. Maryland will incorporate genes for tolerance to fusarium head blight to expand the areas where these varieties can be grown, whereas CIMMYT will provide access to a global network of triticale breeding programs. The final objective of this collaborative network is to accelerate the development of highly productive triticales with excellent breadmaking quality that can be commercialize in multiple regions of the US and the world.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1599	1080	80%
204	1599	1081	20%

Knowledge Area
204 - Plant Product Quality and Utility (Preharvest); 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1599 - Grain crops, general/other;

Field Of Science
1081 - Breeding; 1080 - Genetics;

Keywords

cimmyt

triticale

breeding

genetic mapping

grain quality

Goals / Objectives
The overall objective of this proposal is to enhance our understanding of the genes controlling Breadmaking quality (BMQ) in triticale and to use this information to produce a food-grade triticale for human consumption. The proposed work will bring closer to fruition the vision of Norman Borlaug and many other small grain geneticists and breeders who envisioned triticale as a novel and more productive grain crop for human consumption. This work will be conducted in high yielding, broadly adapted triticale germplasm that could be commercially relevant in multiple grain growing regions of the U.S. and in CIMMYT to reach triticale breeding programs around the world.Our specific objectives are:1) Evaluate grain yield and quality of recently generated triticale lines with unique combinations of five wheat loci with known positive effects on BMQ. We hypothesize that the evaluation of multiple beneficial allele combination in multiple genetic backgrounds will allow us to identify combinations that minimize yield penalties and maximize BMQ.2) Discover the genetic basis of the enhanced loaf volume present in Ukrainian triticale cultivars Timofey (UKR03) and Yaroslava (UKR04) and develop molecular markers to accelerate the deployment of the beneficial alleles. We hypothesize that chromosome regions responsible for the observed difference in loaf volume can be identified by evaluating segregating mapping populations generated from crosses including these Ukrainian lines.3) Deploy and validate the discovered QTL for improved BMQ in breeding populations adapted to different growing regions. Since UKR03 and UKR04 have been used in crosses in our breeding programs, we hypothesize that those breeding populations can be used to select locally adapted lines with improved BMQ and to validate the BMQ QTLs discovered in Objective 2.4) Combine the five wheat loci with known positive effects on BMQ already introgressed in triticale with the newly discovered QTL for improved loaf volume. We hypothesize that by combining these two independent sources of beneficial alleles we can improve triticale BMQ beyond the levels currently achieved in each of the separate germplasm sources.5) Collaborate with the artisan milling and baking community to evaluate and promote triticale for human consumption. We hypothesize that the artisan milling and baking community is more flexible and less averse to innovation than the larger mills, and that it will provide a useful entry point for the triticale into the larger milling and baking industry. Preliminary results presented in section 2.4 are starting to validate this hypothesis.An important and longer-term objective of this work is to strengthen and expand collaborative public sector triticale research and breeding efforts in the U.S. We expect that this initial group of six breeding programs widely distributed across the U.S. and Mexico can showcase the potential of triticale for human consumptions and can grow into a more formal collaborative network for research and improvement of this new crop. Private companies can also benefit from interactions with this nascent network of researchers by learning the potential of this crop, testing the new lines in different environments, expanding screening nurseries and establishing public-private partnerships to promote and improve food-grade triticale for human consumption.

Project Methods
Methods for Objective 1 - Effect of favorable wheat alleles for BMQ introgressed into triticale on quality and grain yield.We developed triticale lines that incorporate different combinations of five different beneficial alleles for BMQ into fall-planted spring triticale cultivars UC-Atrea and UC-Bopak. Specifically, we combined the engineered 'FC2' chromosome that carries the Glu-D1-5+10 and Gli-D1/Glu-D3 loci from bread wheat and removes the detrimental Sec-1 and Sec-3 alleles from rye with the beneficial Glu-A1-2*, Glu-B1-Bx7OE and GPC-B1 alleles from wheat. We will evaluate these lines for BMQ at the California Wheat Commission (CWC) quality lab and at CIMMYT (Obregon) for grain yield and quality evaluation.We will focus on a panel of 64 lines representing a factorial combination of BMQ loci and genetic backgrounds including two separate BC4F5 lines for 32 genotypes. This panel of 64 lines will be evaluated at Davis, CA and at the CIMMYT research facility in Obregon, Mexico in a randomized complete block design (RCBD) with four replications during the 2022-23 and 2023-24 growing season. Grain yield data will be obtained for each of the four replications but quality data will be obtained only four two replications generated by pooling grain from two replications. Grain from the Obregon trials will be evaluated for BMQ at the CIMMYT grain quality lab, and those from Davis at the CWC quality lab. Quality evaluations of grain protein, hardness (SKCS), gluten content (glutomatic) dough stability (mixograph), and loaf volume will follow AACCI methods. Results from this study will provide a quantification of the effects of the individual loci and their epistatic interactions on different BMQ parameters and grain yield and will inform our selection of the loci optimal combinations.Methods for Objective 2 - Determination of the genetic basis of the improved loaf volume in Ukrainian triticales.The Ukrainian winter triticale lines Timofey (UKR03) and Yaroslava (UKR04) have exceptional bread making quality, with the highest loaf volume of any triticale tested over the last three years. We have made multiple crosses to these lines, including spring and winter triticale. To map the chromosome regions controlling loaf volume, we are generating two mapping populations, one in a spring and the other one in a winter background.Spring Population: In 2019, we crossed UKR03 with our highest yielding spring triticale cultivar 'UC-Atrea' and in the F2 and F3 progenies we selected plants with spring growth habit and heading times close to UC-Atrea. We will generate an F5 mapping population that will be evaluated for BMQ at Davis and Obregon in the years 2 and 3 of this proposal and will be genotyped by GBS. We will plant two replications of the complete populations at each site using small plots as experimental units, and pool seeds from the two replications for BMQ analyses. Quality tests will be performed at the CIMMYT and CWC quality labs and will include grain protein, hardness (SKCS), SDS sedimentation, Solvent Retention Capacity (SRC), gluten content (glutomatic), dough stability (mixograph), and loaf volume (baking test).Winter population: In 2019, we crossed UKR03 with winter line RSI336-FC2, which carries the FC2 chromosome and had the best dough stability of any line tested. This population was selected in the F2 and F3 generation in Davenport, WA. We will generate an F5 mapping population which will be evaluated for agronomic traits at all of our winter sites in Tulelake, Washington, Colorado, and Nebraska. In 2024, we will perform a preliminary quality test on each line, including grain protein, hardness (SKCS), dough stability (mixograph), and loaf volume. The remaining seed will be used for yield and quality evaluation trials at Nebraska and Tulelake (2024-25). A second year of evaluations will be performed in NE in 2025-26.Using the QTL(s) for loaf volume and other BMQ parameters identified in both populations, we will develop markers for the region(s) of interest, which will be used to select for this trait in the breeding populations segregating for this trait. As we receive additional quality evaluation on these lines, in the third and fourth year, we will be able to further refine the QTL positions and the selected markers.Methods for Objective 3 - Deployment and validation of loaf volume QTL from the Ukrainian lines in relevant breeding populations.We will advance breeding populations generated from crosses between UKR03 or UKR04 and locally adapted breeding lines in the different winter and spring triticale breeding programs. These populations will provide an opportunity to accelerate the deployment of the mapped BMQ QTL in relevant germplasm for the different regions. During the first year, we will have seven winter and eight spring breeding populations growing as bulked F4 plots distributed across the breeding programs in CA, CO, NE and WA. Each program will pick one hundred heads from the population adapted to their location, which will be planted as headrows for agronomic selection and BMQ evaluation. These lines will be genotyped with the KASP markers flanking the main BMQ QTL and the information will be used both for breeding and validation purposes. In summary, these populations will provide valuable breeding materials with improved BMQ and adapted to the different environments while simultaneously providing a validation of the discovered BMQ QTL in different environments and genetic backgrounds.Methods for Objective 4 - Combine known quality genes with the loaf volume QTL from the Ukrainian lines.We will pursue a marker assisted selection strategy to combine the loaf volume QTL from UKR03 and UKR04 with the five known quality loci present in the BC4F5 lines, designated henceforth as 5Q-lines. We have already crossed the 5Q-lines (spring) with UKR03 and UKR04 (winter) and initiated two selection streams to separate spring and winter growth habits. Among the selected lines, we will use markers to select at least 10 lines carrying the five known favorable alleles. This large number of plants is necessary to have a reasonable chance to find the Ukrainian favorable alleles once the markers for these loci become available by the end of the second year of the project.Because of the large number of alleles, we will perform selection for the quintuple homozygous in two generations. As soon as the markers for the favorable Ukrainian BMQ alleles become available in 2024, we will use them to select plants carrying all five known favorable alleles combined with both the favorable and unfavorable Ukrainian alleles. Seeds from these selected spring lines will be increased, and tested for yield and BMQ at UCD and CIMMYT in the 2025-26 season.Methods for Objective 5 - Engaging artisan millers and bakers in evaluations and market development.We will make 500 lbs of PawoFC2 available to our collaborating artisan mills, who have previously tested this line. In addition, we will provide 200 lbs of UKR04 to these mills to test for the first time. The remaining grain of these two lines will be planted in October 2022, to provide larger volumes of grain for testing and product development.Throughout this proposed work, we will continue to engage with this community, attending their events, learning from their experience, and ensuring they have access to our improved triticales for human consumption. In addition to these established connections, we have received interest and will continue to develop collaborative relationships with the Artisan Grain Collaborative (graincollaborative.com) in Wisconsin, and the Bread Lab at WSU. In addition to these engagements with end user in the U.S., Co-PD Karim Ammar at CIMMYT has multiple collaborators in Ethiopia, India, and other developing countries who are interested to see how our improved triticale can be used for their traditional baked products.

Progress 01/01/24 to 12/31/24

Outputs
Target Audience:The target audiences for this work include researchers, growers, and end users. The researchers include both public and private sector researchers working in triticale genetics and breeding in addition to cereal chemists. The primary grower audiences targeted by this project are the small grain growers of California, Colorado, Washington, Nebraska and Maryland. This project will provide new uses for the triticale grown in these regions and will provide growers with adapted varieties with improved breadmaking quality. The inclusion of the CIMMYT triticale breeding program in this project also expands the audience of this project to a global footprint. Artisan millers and artisan bakers have already indicated interest in triticale for various baked products and will be direct beneficiaries of this research project. Larger commercial mills and bakeries are also a potential targeted audience if the interest in triticale for direct human consumption gets traction. As some of the triticale have unique flavor profiles, the culinary industry and general public will also be included among the targeted audiences to raise general awareness of the product. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The breeding activities that are a part of this grant have given opportunities for undergraduate students interested in plant breeding to gain hands on experience, from crossing to field design, planting, harvesting, and grain quality testing. In the first year of this project, at UC Davis we had one undergraduate student intern paid from this project and another four who participated in various ways. One undergraduate has taken special interest in the development of baking recipes with our triticale and is processing our triticale grain samples at the California Wheat Commission for her senior thesis project. In addition, this project partially supports undergraduate interns at Washington State University, Colorado State University, and the University of Nebraska to help with field management and selections in segregating populations. How have the results been disseminated to communities of interest?Outreach to our audiences of growers and seed industry professionals has taken place at field day events and in some one-on-one field tours. In 2023 this included field days at UC Davis, at the IREC in Tulelake CA and at the Wilke Farm in Davenport, WA. Outreach to our audience of mills and end users has occurred through multiple one-on-one meeting, both in person and virtually. In some cases, we have reached out directly to solicit interest in our triticale, and in other cases they came to us to learn more and/or get samples of grain and flour. Finally, in September 2024 we participated in the Variety Showcase in Portland, Oregon, which is put on by the culinary breeding network. This annual event brings together approximately 500 culinary professionals with breeders displaying interesting new cultivars. We were partnered with Starter Bread Bakery in Portland who produced multiple triticale bread products to share with visitors and we discussed the benefits of triticale and our improvements to its end use quality. Engagement from this event has driven additional sales of flour made from UCT3131 at Camas Country Mill. What do you plan to do during the next reporting period to accomplish the goals?Plans for the coming year are described separately for each specific objective: Obj. 1 - Effect of favorable wheat alleles for BMQ introgressed into triticale on quality and grain yield. In 2025, we will complete the grain quality testing of these lines from trials harvested in 2024 to determine the impact on grain yield and quality associated with each locus. Once this data is complete, we will begin to write the results as a manuscript for publication. Obj. 2 - Determination of the genetic basis of the improved loaf volume in the Ukrainian triticale. The lines which will serve as a genetic mapping population for this objective are currently planted at UC Davis for the 2024-25 growing season for a second year of agronomic evaluation. We will extract DNA from each of these lines and genotype them using genotyping by sequencing (GBS). The GBS reads will be mapped and SNPs called using a new triticale reference genome sequence developed by collaborators in Canada. These SNPs will be used to develop a genetic map. Once we complete the loaf volume measurements of these lines in April 2025, we will be able to map genetic loci responsible for the loaf volume trait and develop molecular markers. Obj. 3 - Deployment and validation of loaf volume QTL from the Ukrainian lines in relevant breeding populations. We will continue to make selections and advance these breeding populations across our breeding locations. In addition, we will make additional crosses of the Ukrainian lines to newly developed breeding lines adapted to various regions of our breeding network. Once we have genetic markers associated with loaf volume, we plan to use them to accelerate the introgression process using marker assisted selection. Obj. 4 - Combine known quality genes with the Ukrainian loaf volume QTL. We will continue making selection and advancing these breeding populations across our breeding locations. For some of these populations, which are planted as F5 headrows in Davis, CA, we will begin to genotype and make selections for the known BMQ loci form Obj. 1. Once we have genetic markers associated with loaf volume, we will be able to select lines varying these loci, in addition to the 5 known BMQ loci. Obj. 5 - Engaging artisan millers and bakers in evaluations and market development. In 2025, we will continue to engage our milling and baker partners to promote the use of our improved triticale. Larger scale milling and baking in 2025 will still be focused on the lines Yaroslava (UKR04) and PawoFC2 (UCT3131), for which we have larger quantities of grain. Camas Country Mill continues to sell flour made from our UCT3131 and we will continue helping them to develop additional interest in and sales of this flour. In addition, we will begin to share smaller samples of the newer lines generated in Obj. 1 and Obj. 2, to select which of these should be moved forward for larger scale testing in subsequent years. Finally, we will again participate in the Variety Showcase to promote our triticale flours and their unique characteristics.

Impacts
What was accomplished under these goals? Accomplishments are described separately for each specific objective: Obj. 1 - Effect of favorable wheat alleles for BMQ introgressed into triticale on quality and grain yield. The 64 lines developed to achieve Aim 1 incorporate different combinations of five different beneficial alleles for BMQ into fall-planted spring triticale cultivars UC-Atrea and UC-Bopak. Specifically, we combined the engineered 'FC2' chromosome that carries the Glu-D1-5+10 and Gli-D1/Glu-D3 loci from bread wheat and removes the detrimental Sec-1 and Sec-3 alleles from rye with the beneficial Glu-A1-2*, Glu-B1al-Bx7OE and GPC-B1 alleles from wheat. During the 2023-24 growing season, these lines were planted in yield trials in Davis, CA and at the CIMMYT experiment station in Obregon, MX. Quality testing of the grain from these trials are still ongoing and are expected to be completed by May 2025. Grain quality testing completed in October 2024 from grain harvested in the 2022-23 growing season showed a significant positive impact on dough stability as measured on a mixograph. The loci Glu-D1, Gli-D1/Glu-D3, and Glu-B1al were associated with an increase in mixing tolerance, respectively, while Glu-A1 and GPC-B1 were not associated with a significant change in mixing tolerance or other quality parameters. On average across all lines, all of the loci, except for Glu-B1al, were associated with a reduction in grain yield, ranging from 6 - 19%. However, there were a few lines carrying all five loci that delivered a grain yield similar to or greater than the recurrent parent (UC-Bopak). On average these lines displayed a 2.9-fold increase in dough stability, with values similar to our high grain quality bread wheat cultivar UC-Central Red. These high yielding lines carrying all five MAS target loci have already been incorporated into numerous crosses in 2024 and will again in 2025. This will increase the frequency of these important alleles and a source of superior lines with improved grain quality in the future. Obj. 2- Determination of the genetic basis of the improved loaf volume in the Ukrainian triticale. To determine the genetic basis of the exceptional loaf volume of the Ukrainian winter triticale lines Timofey (UKR03) and Yaroslava (UKR04), we crossed both to a spring triticale with poor loaf volume to generate a mapping population. In April 2023, from over 900 F4 headrows we selected 264 lines and planted them in a yield trial in Davis, CA in November for the 2023-24 growing season in two replications with the parents and the bread wheat cultivar UC-Central Red repeated throughout the field. In July 2024 this yield trial was harvested and revealed 15 lines (6%) which delivered a grain yield similar to or greater than our triticale cultivar UC-Bopak, while more than 65% of the lines had a grain yield higher than our wheat cultivar, UC-Central Red. Grain samples from each line are currently being tested for quality at the California Wheat Commission and CIMMYT quality laboratories. This testing is expected to be completed by May 2025 and will include grain hardness, milling parameters, mixing tolerance, falling number, SDS sedimentation, alpha amylase activity and loaf volume. This entire population has been planted again in Davis for the 2024-25 growing season. In addition, we have generated additional populations of Timofey (UKR03) and Yaroslava (UKR04) crossed to other winter triticale lines. These populations as planted as F4 head-rows and F5 preliminary yield plots for the 2024-25 growing season in the winter locations. Obj. 3-Deployment and validation of loaf volume QTL from the Ukrainian lines in relevant breeding populations. To facilitate the rapid deployment of the loaf volume trait from the Ukrainian lines, we have made crosses to other breeding material adapted to a wide range of environments. In September 2024 we advanced 31 populations to the F2 selection plots in each relevant breeding location for the 2024-25 season. In addition, we have made selections and advanced populations made in previous years. Obj. 4- Combine known quality genes with the Ukrainian loaf volume QTL. To combine the loaf volume trait from the Ukrainian lines with the other known quality genes, which we have demonstrated have a large impact on mixing tolerance and dough handling, in 2022 we made 10 crosses between the Ukrainian lines and spring lines carrying all five beneficial alleles for BMQ. In 2023, we made 16 additional crosses, some of which were made between F1 plants (4 way cross) and some between the F1 and lines well adapted to particular environments - eg. Spring/ winter, irrigated/ rainfed, etc. These populations are planted in the 2023-24 season as F2 to F3 plots in their optimal environments, depending on the intent of the cross, across our spring and winter breeding locations. In September 2024, we advanced these selections to the F4 single rows in each relevant breeding location for the 2024-25 season. Obj. 5- Engaging artisan millers and bakers in evaluations and market development. To ensure that the materials developed in this project reach the public, we have engaged with multiple craft scale mills and influential artisan bakers, giving them grain and/or flour samples to test the material. In this process we have taken extra time to describe the material and the improvements we have made and hear their experience of how it was to work with, its flavor, and how they would like to incorporate this flour into their operations. In August 2023, we harvested approximately 40,000 lb. of seeds from the line PawoFC2 (UCT3131) and delivered over 36,000 lb. of seeds to Camas Country Mill in October 2023. We also delivered another 150 lbs. of this increase to Grist & Toll. To ensure that we had additional grain to meet possible future demands, we planted another 5-acre increase of PawoFC2 (UCT3131) at the Intermountain Research and Education Center (IREC) for the 2023-24 season. We retained approximately 2000 lb. from the 2023 harvest to be used for additional milling and to be planted by outside growers for either mill in the 2024-25 season. In October 2024, we planted an additional 4 acres of UCT3131 which will be harvested in August 2025 and be made available to interested millers and bakers. In addition to the engagements with these craft scale mills, we have sent samples to multiple influential bakers. This includes Josey Baker who runs 'The Mill', a café and bakery in San Francisco who had positive comments about the flour and are interested to obtain more. In February 2024 we delivered 300 lb. of grain to Josey which was milled out flour and started a new line of bread at his baker he named 'Forage Bread', a 100% whole grain triticale sourdough loaf. In response to strong customer interest, he followed up to buy additional grain from Camas Country Mill multiple times in 2024. Finally, in September 2024, we participated in the Variety Showcase in Portland, OR, which is put on by the culinary breeding network. This annual event brings together approximately 500 culinary professionals with breeders displaying interesting new cultivars. We were partnered with Starter Bread Bakery in Portland who produced multiple triticale bread products to share with visitors and we discussed the benefits of triticale and our improvements to its end use quality. Engagement from this event has driven additional sales of flour made from UCT3131 at Camas Country Mill.

Publications

Progress 01/01/23 to 12/31/23

Outputs
Target Audience:The target audiences for this work includes researchers, growers, and end users. The researchers include both public and private sector researchers working in triticale genetics and breeding in addition to cereal chemists. The primary grower audiences targeted by this project are the small grain growers of California, Colorado, Washington, Nebraska and Maryland. This project will provide new uses for the triticale grown in these regions and will provide growers with adapted varieties with improved breadmaking quality. The inclusion of the CIMMYT triticale breeding program in this project also expands the audience of this project to a global footprint. Artisan millers and artisan bakers have already indicated interest in triticale for various baked products and will be direct beneficiaries of this research project. Larger commercial mills and bakeries are also a potential targeted audience if the interest in triticale for direct human consumption gets traction. As some of the triticale have unique taste profiles, the general public will also be included among the targeted audiences to raise awareness of the product. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The breeding activities that are a part of this grant have given opportunities for undergraduate students interested in plant breeding to gain hands on experience, from crossing to field design, planting, harvesting, and grain quality testing. In the first year of this project, at UC Davis we had one undergraduate student intern paid from this project and another four who participated in various ways. One undergraduate has taken special interest in the development of baking recipes with our triticale and hopes to develop a senior thesis project and work with bakers, which he has worked for in the past, to adopt the use of our improved triticale. In addition, this projectsupports, at least in part, undergraduate inters at Washington State University, Colorado State University, and the University of Nebraska to help with field management and selections in segregating populations How have the results been disseminated to communities of interest?Outreach to our audiences of growers and seed industry professionals has taken place at field day events and in some one-on-one field tours. In 2023 this included field days at UC Davis, at the IREC in Tulelake CA and at the Wilke Farm in Davenport, WA. Outreach to our audience of mills and end users has occurred through multiple one-on-one meeting, both in person and virtually. In some cases we have reached out directly to solicit interest in our triticale, and in other cases they came to us to learn more and/or get samples of grain and flour. Finally, we were invited to contribute flour of our triticale to a public-facing event called Sips and Bites, hosted by the Mondavi Institute at UC Davis. In this event the audience was told about various unique types of grain and four and tasted each. Our triticale flour was a clear winner in the informal audience poll. What do you plan to do during the next reporting period to accomplish the goals?Plans for the coming year are described separately for each specific objective: Obj. 1 - Effect of favorable wheat alleles for BMQ introgressed into triticale on quality and grain yield. In 2024 we will complete the grain quality testing of these lines from trials harvested in 2023 to determine the impact on grain yield and quality associated with each locus. In addition, we will do an additional year agronomic evaluation at CIMMYT and UC Davis in the 2023-24 growing season, with grain quality evaluations to follow. Obj. 2 - Determination of the genetic basis of the improved loaf volume in the Ukrainian triticale. The lines which will serve as a genetic mapping population for this objective are currently planted at UC Davis for the 2023-24 growing season and will be the first opportunity for agronomic evaluation. We will extract DNA from each of these lines and genotype them by genotyping by sequencing (GBS). The GBS reads will be mapped and SNPs called using a new triticale reference sequence being developed by collaborators in Canada, from which we will develop a genetic map. Once we have loaf volume measurements of these lines in late 2024 or early 2025, we will be able to map genetic loci responsible for the loaf volume trait. Obj. 3 - Deployment and validation of loaf volume QTL from the Ukrainian lines in relevant breeding populations. We will continue to make selections and advance these breeding populations across our breeding locations. In addition, we will make additional crosses of the Ukrainian lines to developbreeding lines adapted to various regions of our breeding network. Once we have genetic markers associated with loaf volume, we can use them to select the lines carrying these markers from the advanced populations. Obj. 4 - Combine known quality genes with the Ukrainian loaf volume QTL. We will continue to make selection and advance these breeding populations across our breeding locations. For some of these populations, which are planted as F5 headrows in Davis, CA, we will begin to genotype and make selections for the known BMQ loci form Obj. 1. Once we have genetic markers associated with loaf volume from the Ukrainian lines, we will be able to select lines including these favorable alleles, in addition to the 5 introgressed BMQ loci. Obj. 5 - Engaging artisan millers and bakers in evaluations and market development. In 2024, we will continue to engage our milling and baker partners to promote the use of our triticales improvedfor BMQ. Larger scale milling and baking in 2024 will still be focused on the lines Yaroslava (UKR04) and PawoFC2 (UCT3131), for which we have larger quantities of grain. However, we will begin to share smaller samples of the newer lines generated in Obj. 1 and Obj. 2, to help us select which of these should be moved forward for larger scale testing in subsequent years.

Impacts
What was accomplished under these goals? Obj. 1. Effect of favorable wheat alleles for bread making quality (BMQ) introgressed into triticale on quality and grain yield. The 64 lines developed to achieve Aim 1 incorporate different combinations of five different beneficial alleles for BMQ into fall-planted spring triticale cultivars UC-Atrea and UC-Bopak. Specifically, we combined the engineered 'FC2' chromosome that carries the Glu-D1-5+10 and Gli-D1/Glu-D3 loci from bread wheat and removes the detrimental Sec-1 and Sec-3 alleles from rye with the beneficial Glu-A1-2*, Glu-B1al-Bx7OE and GPC-B1 alleles from wheat. During the 2022-23 growing season, these lines were planted in yield trials in Davis, CA and at the CIMMYT experiment station in Obregon, MX. Quality testing of the grain from these trials are still ongoing and are expected to be completed by April 2024. Grain quality testing completed in March 2023 from grain harvested in the 2021-22 growing season showed a significant positive impact on dough stability as measured on a mixograph. The loci Glu-D1, Gli-D1/Glu-D3, and Glu-B1al were associated with a 27%, 16%, and 24% increase in mixing tolerance, respectively, while Glu-A1 and GPC-B1 were not associated with a significant change in mixing tolerance or other quality parameters. On average across all lines, all of the loci, except for Glu-B1al, were associated with a reduction in grain yield, ranging from 6 - 19%. However, there were a few lines carrying all five loci that delivered a grain yield similar to or greater than the recurrent parent (UC-Bopak). On average these lines displayed a 2.9-fold increase in dough stability, with values similar to our high grain quality bread wheat cultivar UC-Central Red. These lines demonstrate that while on average there may be a tradeoff between the quality loci and grain yield, that some lines will deliver both important traits simultaneously. Obj. 2. Determination of the genetic basis of the improved loaf volume in the Ukrainian triticale. To determine the genetic basis of the exceptional loaf volume of the Ukrainian winter triticale lines Timofey (UKR03) and Yaroslava (UKR04), we crossed both to a spring triticale with poor loaf volume to generate a mapping population. In April 2023, over 900 F4 headrows from these crosses were sown at the Intermountain Research and Extension Center (IREC) in Tulelake, CA. There, we observed a large diversity for many agronomic traits including spike fertility, heading date and maturity time. We selected and harvested grain of 264 of the 907 lines and planted them in a yield trial in Davis, CA in November for the 2023-24 growing season. In addition, we have generated additional populations of Timofey (UKR03) and Yaroslava (UKR04) crossed to other winter triticale lines. These populations are in the F2 to F3 stage, and were planted in the 2022-23 growing season at each of our winter locations, including Lind and Davenport, WA, Akron, CO, and Sidney, NE. These populations were harvested in August 2023 and planted at the F3 to F4 stage for the 2023-24 growing season in all of the same winter locations. Obj. 3. Deployment and validation of loaf volume QTL from the Ukrainian lines in relevant breeding populations. To facilitate the rapid deployment of the loaf volume trait from the Ukrainian lines, we have made crosses to other breeding material adapted to a wide range of environments. In 2023, we made 31 new crosses which included Timofey (UKR03) or Yaroslava (UKR04), which are planted for the 2023-24 growing season as F2 selection plots in the relevant breeding location. In addition, we have continued to make selections and advance in additional populations made in previous years. Obj. 4. Combine known quality genes with the Ukrainian loaf volume QTL. To combine the loaf volume trait from the Ukrainian lines with the other known quality genes, which we have demonstrated have a large impact on mixing tolerance and dough handling, in 2022 we made 10 crosses between the Ukrainian lines and spring lines carrying all five beneficial alleles for BMQ. In 2023, we made and additional 16 crosses, some of which were made between F1 plants (4 way cross) and some between the F1 and lines well adapted to particular environments - eg. Spring/ winter, irrigated/ rainfed, etc. These populations are planted in the 2023-24 season as F2 to F3 plots in their optimal environments, depending on the intent of the cross, across our spring and winter breeding locations. Obj. 5. Engaging artisan millers and bakers in evaluations and market development. To ensure that there is a group of end users eager to work with the improved lines that will results from this research, we have engaged with multiple craft scale mills and influential artisan bakers, giving them grain and/or flour samples of various sizes to test the differnt lines. In this process we have taken extra time to describe the material and the improvements we have made and hear their experience of how it was to work with these lines, theirflavor, and how they would like to incorporate the improved triticale flour into their operations. To have enough grain for these end user evaluations, in the 2021-22 growing season we planted a grain increase of our two lines with the best grain yield and quality, and which were preferred by end users from test in previous years. In September 2022 we harvested approximately 400 lbs. of the line Yaroslava (UKR04) and 900 lbs. of PawoFC2 (UCT3131) at the IREC station in Tulelake, CA. To ensure that there would be a larger quantity of grain to meet possible demand, we then planted for the 2022-23 season 4.5 acres of PawoFC2 (UCT3131) at IREC, which had been slightly preferred for is superior dough handling. In early 2023 we gave approximately 150 lbs. of each line to each of two craft scale mills, Grist & Toll in Pasadena, CA and Camas Country Mill in Eugene, OR. Each mill produced flour of these lines and gave samples to their customers to test out in various recipes and report back about their interest in purchasing more in the future. Both mills receive very positive feedback from their customers, who used the flour to make a range of products from sourdough bread to muffins, cookies, and crackers. They were most excited by the flavor of both triticale flours, which they compared to a rye-wheat flour blend, but with much better functionality in the bakery. In August 2023, we harvested approximately 40,000 lbs. of grain from our 4.5 acre increase of PawoFC2 (UCT3131). Encourage by the positive feedback they had received, Camas Country Mill requested to pick up as much of this grain as we would release to them, ultimately taking just over 36,000 lbs on October 2023. We also delivered another 150 lbs. of this increase to Grist & Toll. To ensure that we had additional grain to meet possible future demand, we planted another 5-acre increase of PawoFC2 (UCT3131) at IREC for the 2023-24 season, and have retained approximately 2000 lbs from the 2023 harvest to be used for additional milling or to be planted by outside growers for either mill in the 2024-25 season. In addition to engagements with these craft scale mills, we have sent samples to multiple influential bakers. This includes Josey Baker who runs 'The Mill', a café and bakery in San Francisco, and Patch Troffel and Dan Barber (multi-winner of James Beard award) with Blue Hill at Stone Barns in New York. Both groups had positive comments about the flour and are interested to obtain more for additional experimentation and to possibly develop production products using this flour. With all of this engagement of craft scale mills and artisan bakers, our collaborators at Bay State Milling, a medium scale food ingredient company, became interested in testing our new triticale. In late 2023 we shipped 20 lbs each of three of our improved triticale lines to their R&D bakery for evaluation.

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