Source: NORTH DAKOTA STATE UNIV submitted to
ENRICHING AND UNDERSTANDING THE WHEAT GENOME BY INDUCING SECONDARY HOMOEOLOGOUS RECOMBINATION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
EXTENDED
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1018529
Grant No.
2019-67013-29164
Project No.
ND05068
Proposal No.
2018-06269
Multistate No.
(N/A)
Program Code
A1141
Project Start Date
Mar 1, 2019
Project End Date
Feb 28, 2023
Grant Year
2019
Project Director
Cai, X.
Recipient Organization
NORTH DAKOTA STATE UNIV
1310 BOLLEY DR
FARGO,ND 58105-5750
Performing Department
Plant Sciences
Non Technical Summary
Wheat is a major food crop in the US. Wheat production has been continually threatened by various biological and environmental stresses. There is a constant need to discover and deploy new genes into wheat to overcome the emerging threats. However, the narrow genetic variability of the wheat genome has made this a challenging task. It is essential to diversify the wheat genome and expand the gene pool for wheat breeding. Here, we propose to enrich and understand the wheat B genome, the ancestor of which remains obscure, by genomics-enabled chromosome engineering. Genome-wide engineering will be performed to introduce novel disease resistance and salt/waterlogging tolerance genes from wheat-related wild grasses into wheat by inducing gene exchange between wheat and the wild grasses. In addition, we will develop an effective chromosome engineering-based pipeline of gene flow from wild grasses into wheat by taking advantage of the recent advances in genomics. This will improve the efficacy and throughput of chromosome engineering in wheat breeding. Meanwhile, this research will enhance understanding of the wheat genome, which is essential for the genetic improvement of wheat. The outcomes of this project will include wild grass-derived wheat germplasm that can be utilized immediately for the development of superior wheat varieties and a unique chromosome engineering-based genomic framework useful in wheat genome studies. Overall, this research will strengthen the defense of US wheats against the biological and environmental stresses and sustain/improve wheat production under the emerging threats. This project addresses the Program Area Priority "Plant Breeding for Agricultural Production".
Animal Health Component
0%
Research Effort Categories
Basic
30%
Applied
70%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20215491081100%
Knowledge Area
202 - Plant Genetic Resources;

Subject Of Investigation
1549 - Wheat, general/other;

Field Of Science
1081 - Breeding;
Goals / Objectives
Wheat is a major food grain source for humans in the US and worldwide. Wheat production has been continually threatened by various biological and environmental stresses, such as new diseases, insects, and climate variability/change. There is a constant need to enhance the genetic potential of wheat in sustaining and improving wheat production under emerging threats. In recent years, the genetic gain for wheat production has declined due to the draining of the primary gene pool in wheat breeding. The limited genetic variability of the wheat genome has increasingly become a bottleneck for wheat improvement. However, wheat has a huge secondary and tertiary gene pool (i.e. wheat-related wild species) with tremendous genetic variability, representing an invaluable gene source for wheat breeding. The long-term goal of this project are to enrich the wheat genome for wheat improvement by harnessing the genetic diversity of wheat-related wild grasses and to understand the wheat genome by chromosome manipulation.Over the last decade, we have performed genomics-enabled chromosome engineering to harness genetic variability from wild species for enriching the gene content of the wheat genome. To date, we have produced a large set of wheat germplasm materials (n>1,000) by introducing the genes from goatgrass (Aegilops speltoides, genome SS) and tall wheatgrass (Thinopyrum elongatum, genome EE) into wheat through the genomics-enabled chromosome engineering. In addition, these wheat materials are invaluable genetic resources for genome studies in wheat and its relatives. Many of the wild grass-derived wheat germplasm materials contain the favorable genes useful in wheat breeding (e.g. resistance/tolerance genes for wheat diseases and environmental stresses). However, some of the wheat materials contain the undesired grass genes we do not want, such as late maturity, tall stature, and undesired end-use quality, in addition to the favorable genes. The wild grass-derived undesirable genes need to be removed from the wheat materials to improve their utilities in wheat breeding by chromosome engineering. This chromosome engineering-based alien gene introgression will enrich the genetic composition of the wheat genome and expand its genetic variability. Meanwhile, this work will lead to the development of invaluable genetic resources useful in the genome studies of wheat and its relatives. Following are the specific objectives of this research project and a brief description for each of the objectives.Objective 1. Enhance B-S and B-E recombination toward proximal chromosomal regions and reduce alien segments in the favorable recombinants (Year 1-3)We will produce wheat materials that contain the genes of interest on the reduced wild grass chromosome segments without deleterious genes under this objective. The wild grass-derived genes targeted in this project include those for resistance to the rust, tan spot, Stagonospora nodorum blotch (SNB), and Fusarium head blight diseases and for tolerance to salt and waterlogging. We anticipate developing breeder-friendly wheat germplasm containing the wild grass-derived resistance/tolerance genes for the development of superior varieties. This will make the wild grass genes usable in wheat breeding and strengthen the defense of the U.S. wheats against the diseases and environmental stresses.Objective 2. Construct composite bin maps of wheat B-genome chromosomes at a relatively high resolution (Year 2-3)The wheat materials developed under Objective 1 dissect the large and complex genome of wheat and are extremely useful in wheat genome studies. We will use those materials to partition the wheat genome and position the genes and other DNA sequences in the wheat genome. This will lead to the construction of a unique framework for the genome studies in wheat and its relatives.Objective 3. Remove the ph1b mutant allele from the B-S and B-E recombinants and develop homozygous recombinant lines for germplasm development and genome studies (Year 2-3)The mutant gene (i.e. ph1b mutant) used for chromosome engineering in this study will be removed from the germplasm materials because it causes genetic instability. The genetically-stable wheat germplasm homozygous for the wild grass-derived genes of interest will be developed under this objective. This will make the wheat germplasm materials ready for variety development in wheat breeding. Objective 4. Evaluate the B-S and B-E recombinant lines for the traits of interest and develop breeding-ready germplasm in the adapted wheat backgrounds (Year 3-4)We will evaluate the wheat germplasm materials for the traits targeted in this study and select the germplasm that contain the gene of interest, but no deleterious genes. Meanwhile, we will incorporate the wild grass-derived favorable genes into the adapted wheat genotypes to develop elite germplasm lines for variety development in wheat breeding.Ultimately, this research will boost the genetic potential of wheat in sustaining and improving U.S. wheat production under emerging biological and environmental threats, and facilitate chromosome engineering-based genome studies in wheat and its relatives. In addition, we will optimize the genomics-enabled chromosome engineering technology for genome-wide manipulation and characterization in wheat and its relatives. This will further improve the efficacy and throughput of chromosome engineering in wheat breeding and genome study.
Project Methods
Recent advances in genomics have provided a great opportunity to improve the efficacy and throughput of chromosome engineering in wheat breeding. We will take the advantage of the genomic technologies and resources currently available in wheat and its relatives to develop an effective genomics-enabled chromosome engineering procedure for alien gene introgression and genome study in wheat and its relatives. Following are the brief description of the methods/techniques we will develop and use to accomplish each of the objectives.Objective 1. Enhance B-S and B-E recombination toward proximal chromosomal regions and reduce alien segments in the favorable recombinants (Year 1-3)We will use the ph1b mutant to introduce the genes of interest from wild grasses into wheat. Multiple artificial crosses will be involved in this gene transfer process. DNA markers and genomic in situ hybridization (GISH) will be used to assist selection of the wild grass-derived genes/DNA in the progeny of the crosses for the development of the wheat germplasm containing the genes of interest.Objective 2. Construct composite bin maps of wheat B-genome chromosomes at a relatively high resolution (Year 2-3)The wheat germplasm materials developed under Objective 1 will be genotyped by the high-throughput wheat 90K SNP assay. This will physically partition the wheat B-genome chromosomes and lead to the construction of a unique chromosome engineering-based framework (i.e. composite bin map) for the wheat B genome.Objective 3. Remove the ph1b mutant allele from the B-S and B-E recombinants and develop homozygous recombinant lines for germplasm development and genome studies (Year 2-3)Recently, we have developed new diagnostic DNA markers specific for the ph1b mutant. We will use those DNA markers to assist selection of the germplasm materials without the ph1b mutant gene. Meanwhile, we will develop the germplasm materials homozygous for the wild grass-derived genes/DNA using the DNA markers specific for the wild grass chromosomes. Ultimately, we expect to develop the genetically-stabilized wheat germplasm lines containing wild grass-derived genes of interest for breeding use and maintenance.Objective 4. Evaluate the B-S and B-E recombinant lines for the traits of interest and develop breeding-ready germplasm in the adapted wheat backgrounds (Year 3-4)The genetically-stabilized wheat germplasm lines developed under the previous objectives will be evaluated for the traits targeted in this project, including resistance to the rust, tan spot, Stagonospora nodorum blotch (SNB), and Fusarium head blight diseases and tolerance to salt and waterlogging. Also, we will evaluate those germplasm lines for overall agronomic performance and select the best lines for the development of elite breeding-ready germplasm in the adapted wheat backgrounds. The wild grass chromosome-specific DNA markers will be used to assist selection in the germplasm development processes.

Progress 03/01/19 to 04/13/21

Outputs
Target Audience:There is a shortage of the scientists/researchers trained in plant molecular cytogenetics in the US and worldwide. One graduate student, one postdoctoral researcher, and three undergraduate students have participated in this research project. They have received extensive training in classical and molecular cytogenetic techniques and genomics-enabled chromosome engineering technology. In addition, they have learned the new pipeline we have developed in chromosomeengineering and genome manipulation and characterization in this project. Also, we have brought the research materials and results, especially in chromosome engineering and genome enrichment, we have obtained in this project into the graduate cytogenetics class (PLSC 741). The work we have done in this project has provided a great opportunity to graduate students to learn genomics-enabled chromosome engieering and alien gene introgression for plant improvement and genome study. Changes/Problems:The PI is leaving his current position at North Dakota State University (NDSU) and taking a new Research Geneticist position with USDA-ARS in Lincoln, NE. This research grant is in the process of transferringfrom NDSU to USDA-ARS. This is why the PI is submitting thisfinal report of this project via NDSU although this grant ends on February 28, 2023. Theresearch objectives will be implemented and accomplished in the same way as proposed. No significant changes will be made with this research project at this moment. What opportunities for training and professional development has the project provided?One postdoctoral research fellow, one graduate student, three undergraduate students, and one research specialist have participated in this research project. They have received extensive training in molecular cytogenetics and genomics-enabledchromosome engineering from PI and collaborators. Also, PI, postdoc, research specialist, and graduate student have presented and communicated the results of this project with the colleagues in the Plant and Animal Genome conference (San Diego, CA).One Ph.D. student who worked on this project graduated in 2020.? How have the results been disseminated to communities of interest?Research results and technologies of this project have been presented in the international scientific conference (the Plant and Animal Genome conference ). In addition, this work has been presented to the commodity groups, such as ND Wheat Commission, to help wheat growers understand the significance and impact of this research on wheat production. Also, we have used some of the research materials and results in the genetics/cytogenetics classes to enhance students understanding of chromosome engineering and its application in crop improvement. To date, four refereed papers and five posters about the research results from this project have been published and presentedin the international scientific journals and international scientific conference. PI was invited to give an oral presentation of this research in the Joint 2019 National Association of Plant Breeders Meeting and NIFA-AFRI PD Meeting, August 25-29, 2019, Georgia. Additional manuscripts about this work are under preparation. What do you plan to do during the next reporting period to accomplish the goals?Significant progress has been made toward the research goals of this project. We will continue making efforts toward the research goals in the following areas during the next reporting period: 1. Generate and recover new recombinants involving homoeologous B-S and B-E chromosome pairs in all groups using chromosome-specific DNA markers and GISH/FISH; 2. Induce secondary and tertiary homoeologous recombination to develop new recombinants toward the proximal chromosomal regions and involving smaller alien chromosomal segments; 3. Reduce the alien chromosomal segments containing the genes targeted in this project and physically map the genes of interest; 4. Develop user-friendly DNA markers diagnostic for the genes of interest and perform marker-assisted introgression of the wild species-derived genes into adapted wheat genotypes for breeding-ready germplasm development; 5. Genotype the recombinants using high-throughput SNP arrays and construct physical maps of wheat B-genome chromosomes; 6. Develop homozygous recombinant lines for trait evaluation and germplasm development; 7. Train the postdoctoral research fellow and graduate/undergraduate students; 8. Publish and present research results.

Impacts
What was accomplished under these goals? The limited genetic variability of the wheat genome has increasingly become a bottleneck for wheat improvement. It is in an urgent need to enrich, diversify, and understand the complex polyploid genome of wheat. The primary goals of this project are to enrich the gene pool of wheat and to develop a unique physical framework of the wheat genome by inducing meiotic recombination of wheat B-genome chromosomes with their homoeologous counterparts in the wild species Aegilops speltoides and Thinopyrum elongatum. These two wild species contain the genes for resistance to major wheat diseases and tolerance to waterlogging and salt. We have produced a large set of homoeologous recombinants with diverse genetic makeup for germplasm development and physical mapping of the wheat B genome. Wheat germplasm containing the wild species-derived favorable genes have been developed. Also, we have developed chromosome-specific DNA markers and genome-specific FISH (fluorescent in situ hybridization) probes to increase the efficacy and throughput of homoeologous recombination-based chromosome engineering. Thus, this work bridges gene flow from the wild species into wheat and diversifies the wheat genome. Objective 1. Enhance B-S and B-E recombination toward proximal chromosomal regions and reduce alien segments in the favorable recombinants (Year 1-3) We have recovered and characterized 141 new recombinants for the homoeologous pairs 1B-1S and 1D-1S from the primary and secondary homoeologous recombination populations. A total of 1,408 individuals from 123 recombination populations were screened to recover new recombinants using chromosome-specific KASP markers. Marker-assisted selection of recombinants were verified by multi-color FISH. The new recombinants involve chromosomes 1B, 1D, and 1S, and contain reduced alien segments. Overall, the meiotic recombination has been shifted toward the proximal chromosome regions in the secondary recombination cycle. Multiple crosses have been made to induce secondary and tertiary recombination toward the proximal regions of other B-E and B-S homoeologous pairs. In addition, we have induced additional recombination of the 2B-2S and 7B-7E recombinants containing resistance genes to stem rust, tan spot, Stagonospora nodorum blotch (SNB), and Fusarium head blight (FHB) diseases to eliminate deleterious genes. New recombinants with reduced alien segments containing the genes of interest have been developed and will be phenotyped. Also, we have been transferring the Ae. speltoides- and Th. elongatum-derived disease resistance genes to the adapted common and durum wheat backgrounds for the development of breeding-ready germplasm. The ph1b mutants we developed in the adapted US wheats have been utilized to induce additional rounds of recombination, producing alien introgression lines in the adapted US wheat backgrounds. We have developed multiple SNP-based KASP and STARP markers defining the distal, pericentromeric, and middle regions for the homoeologous pairs 1B-1S, 1B-1E, 2B-2S, 2B-2E, 3B-3E, 6B-6S, 6B-6E, 7B-7S, and 7B-7E. They have been used to pre-screen the recombination populations for homoeologous recombinants in a relatively high throughput, enabling large-scale recovery of meiotic recombination products. We performed shallow sequencing of the Ae. speltoides (SS) and Th. elongatum (EE) genomes and comparative analysis of their genome sequences to identify genome-specific repetitive DNA sequences for FISH probe development. To date, we have developed three S genome-specific and three E genome-specific FISH probes. They are extremely useful to specifically paint S- and E-genome chromosomes in multi-color FISH analysis. These probes have been used to verify the marker-assisted selection of the recombinants and to physically map the meiotic homoeologous recombination breakpoints. Objective 2. Construct composite bin maps of wheat B-genome chromosomes at a relatively high resolution (Year 2-3) Composite bin maps have been constructed for wheat chromosomes 2B, 3B, and 7B by delineating 2B-2S, 2B-2E, 3B-3E, 7B-7S, and 7B-7E recombinants using wheat 90K SNP arrays and FGISH (fluorescent genomic in situ hybridization). The composite bin map of chromosome 1B is under construction based on 1B-1S and 1B-1E recombination. These composite bin maps illustrate the homoeologous recombination breakpoints, distribution and frequency of recombination events along the entire chromosome, bin size, and SNPs assigned to each of the bins on a chromosome. This homoeologous recombination-based mapping physically dissects wheat chromosomes and their homoeologues in wild species, and provides a unique physical framework for further genome studies in wheat and its relatives. We will construct composite bin maps for wheat B-genome chromosomes 4B, 5B, and 6B and their homoeologues in Ae. speltoides and Th. elongatum following the procedure we have developed. Objective 3. Remove the ph1b mutant allele from the B-S and B-E recombinants and develop homozygous recombinant lines for germplasm development and genome studies (Year 2-3) The SNP-derived KASP and STARP markers diagnostic for Ph1 and ph1b deletion have been used to eliminates ph1b deletion from the recombinants, which could not be done by the previously-reported markers. We have identified over 80% self-pollinated progeny of the primary heterozygous recombinants as Ph1Ph1 or Ph1ph1b by the markers. Several dozens of genetically stabilized homozygous recombinant lines without ph1b deletion have developed for the homoeologous pairs 1B-1S, 2B-2S, 2B-2E, 3B-3S, 7B-7S, and 7B-7E. Meanwhile, we have pre-screened the self-pollinated progeny of the original heterozygous recombinants for homozygous recombinants using chromosome-specific PACE or STARP markers. New recombinants have been detected in addition to the original ones from the self-pollinated progeny. The marker-based selection of homozygous recombinants has been verified by FGISH or multi-color FISH. A total of 256 new homozygous recombinant lines involving homoeologous pairs 1B-1S and 1D-1S have been developed. Sixteen of them were derived from 1D-1S recombination. The homozygous recombinant lines will be genotyped using 90K SNP arrays, and used in the composite bin mapping. The same procedure has been used to recover homozygous recombinants without ph1b deletion from self-pollinated progeny of other original B-S and B-E recombinants. Objective 4. Evaluate the B-S and B-E recombinant lines for the traits of interest and develop breeding-ready germplasm in the adapted wheat backgrounds (Year 3-4) We have delimited the genes for resistance to stem rust, tan spot, and SNB diseases to the distal region on the short arm of Ae. speltoides chromosome 2S, and the gene for FHB resistance (Fhb7) to the distal region on the long arm of Th. elongatum chromosome 7E. These resistance genes have been incorporated into the wheat genome by induced 2B-2S and 7B-7E recombination. We have developed STARP and KASP markers tagging the genes on the alien chromosome segments and have been engineering the recombinant chromosomes to reduce the alien segments containing the resistance genes and to minimizing potential linkage drag associated with the genes. We have been deploying the wild species-derived resistance genes into adapted US wheat genotypes for breeding-ready germplasm development using marker-assisted backcrossing breeding scheme. They will be released to the wheat breeding programs for superior variety development. Also, we have been developing homozygous recombinant lines involving other homoeologous pairs and evaluating the traits of interest for gene identification and mapping. The newly-identified genes will be introgressed into adapted wheat backgrounds for germplasm development.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhang, M., Zhang, W., Zhu, X., Sun, Q., Chao, S., Yan, C., Xu, S.S., Fiedler, J. D., and Cai, X. 2020. Dissection and physical mapping of wheat chromosome 7B by inducing meiotic recombination with its homoeologues in Aegilops speltoides and Thinopyrum elongatum. Theor Appl Genet 133:34553467. https://doi.org/10.1007/s00122-020-03680-3
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhang, M., Zhang, W., Zhu, X., Sun, Q., Chao, S., Yan, C., Xu, S.S., Fiedler, J. D., and Cai, X. 2020. Partitioning and mapping of wheat chromosome 3B and its homoeologue in Thinopyrum elongatum by homoeologous recombination. Theor Appl Genet 133:12771289.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Talukder, Z. I., Underwood, W., Misar, C. G., Seiler, G. J., Liu, Y., Li, X., Cai, X., and Qi, L. 2021. Unraveling the Sclerotinia basal stalk rot resistance derived from wild Helianthus argophyllus using a high-density SNP linkage map. Front Plant Sci. 2021;11:617920. Published 2021 Feb 3. doi:10.3389/fpls.2020.617920
  • Type: Journal Articles Status: Accepted Year Published: 2021 Citation: Gill, B. K., Klindworth, D. L., Rouse, M. N., Zhang, J., Zhang, Q., Sharma, J. S., Chu, C., Long, Y., Chao, S., Olivera, P.D., Friesen, T. L., Zhong, S. , Jin, Y., Faris, J. D., Fiedler, J. D., Elias, M. E., Liu, S., Cai, X., and Xu, S. S. 2021. Function and evolution of allelic variation of Sr13 conferring resistance to stem rust in tetraploid wheat (Triticum turgidum L.). The Plant J.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Zhang, M., Zhang, W., Zhu, X., Sun, Q., Chao, S., Yan, C., Fiedler, J., Xu, S. S., and Cai, X. 2020. Partitioning and physical mapping of wheat chromosome 3B and its homoeologue 3E in Thinopyrum elongatum by inducing homoeologous recombination. Plant and Animal Genome XXVIII Conference, San Diego, CA. January 11-15, 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Zhang, W., Zhang, M., Zhu, X., Ren, S., Fiedler, J., Xu, S. S., and Cai, X. 2020. Dissection and cytological mapping of an Ae. speltoides-originated gene for stunted growth in wheat. Plant and Animal Genome XXVIII Conference, San Diego, CA. January 11-15, 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Danilova, T. V., Zhang, W., Zhang, M., Zhu, X., Poland, J., Fiedler, J., Friebe, B., Cai, X. 2020. Development and application of genome-specific SNP markers for tracing alien introgressions in polyploid wheat genome. Plant and Animal Genome XXVIII Conference, San Diego, CA. January 11-15, 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Zhang, J., Long, Y., Gill1, B. K., Sharma, J. S., Zhang, Q., Klindworth, D. L., Cai, X., Friesen, T. L., Faris, J. D., Xu, S. S. 2020. Development and validation of semi-thermal asymmetric reverse PCR markers for Ug99-effective stem rust resistance genes in wheat. Plant and Animal Genome XXVIII Conference, San Diego, CA. January 11-15, 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Ren, S., Zhu, X., Leng, Y., Zhang, W., Talukder, Z., Zhong, S., Fiedler, J., Qi, L., and Cai, X. 2020. Toward a better understanding of the hexaploid wheat-derived Fusarium head blight resistance in durum wheat. 2020 National Fusarium Head Blight Forum, Virtual Forum via Zoom, December 7-11, 2020.


Progress 03/01/19 to 02/29/20

Outputs
Target Audience:There is a shortage of the scientists/researchers trained in plant molecular cytogenetics in the US and worldwide. Threegraduate students, onepostdoctoral researcher, and twoundergraduate students have participated in this researchproject. They have received extensive training inclassical and molecular cytogenetic techniques and high-throughputgenotyping technology. In addition, they have learned new technologies we have developed in chromosomeengineering and genome manipulation and characterization in this project. Also, we have brought the research materials andresults, especially in chromosome engineering and genome enrichment, we have obtained in this project into undergraduategenetics classes (BIO/BOT/ZOO/PLSC 315 and BIO/BOT/ZOO/PLSC 315 Lab) to arouse students' interest in science. Also,we have applied the results and information generated in this project in the graduate cytogenetics course (PLSC 741). Thework we have done in this project has provided a great opportunity to graduate students to learn genomics-enabledchromosome engieering and alien gene introgression for plant improvement. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One postdoctoral research fellow, twograduate students, twoundergraduate students, and oneresearch specialist have participated in this research project. They have received extensive training in molecularcytogenetics and high-throughput genotyping from PIand collaborators. Also, PI,postdoc, and graduatestudents have presented the results of this project in the Plant and Animal Genome conference (San Diego, CA),the First International Wheat Congress (Saskatoon, Canada),and the NIFA Annual PD meetings. How have the results been disseminated to communities of interest?Research results and technologiesof this project have been presented in the international scientific conferences (the Plant andAnimal Genome conference and 1st International Wheat Congress), theNIFA Annual PD meeting, and to the commodity groups, such as ND WheatCommission, to help wheat growers understand the significance and impact of this research on wheat production. Also, wehave used some of the research materials and results in the genetics/cytogenetics classes to enhance studentsunderstanding of chromosome engineering and its application in crop improvement. Twopapers and one book chapterabout the research results from this project have been published in the international scientific journals and plant breedingbook. Additionalmanuscripts about this work are under preparation. What do you plan to do during the next reporting period to accomplish the goals?Significant progress has been made toward the research goals of thisproject. We will continue making efforts toward the research goals in the followingareas during the next reporting period: 1. Generate and recovernew recombinants involving homoeologous B-S and B-E chromosome pairs in all groups using chromosome-specific DNA markers andGISH; 2. Induce secondary and tertiaryhomoeologous recombination to developnew recombinants toward theproximal chromosomal regions and involving smaller alien chromosomal segments; 3. Reduce the alien chromosomal segments containing the genes targeted in this project and physically map the genes of interest; 4. Develop user-friendly DNA markers diagnostic for the genes of interest and perform marker-assisted introgression of the wild species-derived genes into adapted wheat genotypes for breeding-ready germplasm development; 5. Genotype the recombinants using high-throughput SNP arrays and construct physical maps of wheat B-genome chromosomes; 6. Develop homozygous recombinant lines for trait evaluation and germplasm development; 7. Train the postdoctoral research fellow and graduate/undergraduate students; 8. Publish and present research results.

Impacts
What was accomplished under these goals? Wheat has limited genetic variability due to the polyploid origin of its genome, which has become a bottleneck of wheat breeding.There is an urgent need to enrich, diversify, and understand the complex polyploid genome of wheat.The primary goals of this research project are to enrich the gene pool of wheat and to develop a physical framework for wheat genome studies by performing meiotic homoeologous recombination-based genome mapping and gene introgression from the wild speciesAegilops speltoidesandThinopyrum elongatuminto wheat.These two wild species contain the genes for resistance to major wheat diseases and tolerance to waterlogging and salt.We have produced a large set of wheat introgression lines with diverse genetic makeup for germplasm development and physical mapping of the wheat B genome.Wheat germplasm containing the wild species-derived favorable genes have been developed.Also, we have developed chromosome-specific DNA markers to increase the efficacy and throughput of homoeologous recombination-based chromosome engineering in this project.Thus, this work bridges gene flow from the wild species into wheat and diversifies the wheat genome by harnessing genetic diversity of the wild species. Objective 1.Enhance B-S and B-E recombination toward proximal chromosomal regions and reduce alien segments in the favorable recombinants (Year 1-3) We have developed 112 new recombinants for the homoeologous pairs 1B-1S, 6B-6S, 6B-6E, and 7B-7S in the first year of this project.Also, we have developed 74 new homozygous recombinant lines for the homoeologous pairs 1B-1S, 6B-6S, and 6B-6E.Thirty-two new chromosome-specific PACE (PCR Allelic Competitive Extension) and STARP(semi-thermal asymmetric reverse PCR)markers have been developed to tag the distal regions on both arms and pericentromeric regions of the B-E and B-S homoeologous pairs.They have been extremely useful in high-throughput pre-screening of the large recombination populations for homoeologous recombinants.In addition, we have made multiple crosses to induce secondary and tertiary recombination toward the proximal regions of the B-E and B-S homoeologous pairs.The secondary and tertiary recombination populations will be pre-screened by the chromosome-specific markers to recover new recombinants toward the proximal chromosomal regions. We have been inducing additional recombination of the 2B-2S and 7B-7E recombinants containing resistance genes to stem rust, tan spot, Stagonospora nodorum blotch (SNB), and Fusarium head blight (FHB) diseases to eliminate deleterious genes, such as the gene for stunted growth on the chromosome 2S segment of the 2B-2S recombinant.Also, we have been transferring theAe. speltoides- andTh. elongatum-derived disease resistance genes to the adapted common and durum wheat backgrounds for the development of breeding-ready germplasm.Theph1bmutants we developed in the adapted US wheats in another NIFA-supported project have been utilized to induce additional rounds of recombination in this project, leading to the production of alien introgression lines in the adapted US wheat backgrounds. In addition, we have identifiedAe. speltoidesS genome-specific repeats to develop FISH (fluorescentin situhybridization) probes specific for the S genome by shallow genome sequencing.The S genome-specific repeats work more efficiently than total genomic DNA as a FISH probe for recombinant chromosome analysis.Also, we have been identifyingTh. elongatumE genome-specific repeats for FISH probe development using the same pipeline.This will dramatically improve the efficacy of FISH in homoeologous recombination-based genome study and alien introgression. Objective 2.Construct composite bin maps of wheat B-genome chromosomes at a relatively high resolution (Year 2-3) We have constructed composite bin maps for wheat chromosomes 2B, 3B, and 7B by delineating 2B-2S, 2B-2E, 3B-3E, 7B-7S, and 7B-7E recombinants using wheat 90K SNP arrays and FGISH (fluorescent genomicin situhybridization).The composite bin maps illustrate homoeologous recombination breakpoints and frequencies along the entire chromosome, bin size, and SNPs assigned to each of the bins on a chromosome.This homoeologous recombination-based mapping work physically dissects wheat chromosomes and their homoeologues in wild species, and provides a unique physical framework for further genome studies in wheat and its relatives.Development of additional recombinants for the B-S and B-E homoeologous pairs has improved the resolution of the composite bin maps.We will construct composite bin maps for other wheat B-genome chromosomes and their homoeologues inAe. speltoidesandTh. elongatumfollowing the procedure we have developed. Objective 3.Remove theph1bmutant allele from the B-S and B-E recombinants and develop homozygous recombinant lines for germplasm development and genome studies (Year 2-3) We developed co-dominant SNP-based PACE markers diagnostic for thePh1allele andph1bdeletion.These PACE markers can differentiate the genotypesPh1Ph1,Ph1ph1b, andph1bph1bfrom each other, which could not be done by the previously-reported markers.They are extremely useful in selecting recombinant lines without aph1bdeletion (i.e.Ph1Ph1) immediately in the self-pollinated progeny of the original hemizygous (Ph1ph1b) recombinants.Meanwhile, we have selected homozygous recombinant lines from the self-pollinated progeny of the original heterozygous recombinants using chromosome-specific PACE or STARP markers.These two sets of markers have permitted us to select homozygous recombinant lines without aph1bdeletion in a significantly improved throughput.Homozygosity of the selected recombinants has been verified by FGISH.To date, we have developed 74 new homozygous recombinant lines for the homoeologous pairs 1B-1S, 6B-6S, and 6B-6E.We will continue selecting homozygous recombinant lines withoutph1bdeletion from self-pollinated progeny of other original B-S and B-E recombinants by marker and GISH/FISH analysis. Objective 4.Evaluate the B-S and B-E recombinant lines for the traits of interest and develop breeding-ready germplasm in the adapted wheat backgrounds (Year 3-4) We have identified and mapped resistance genes for stem rust, tan spot, and SNB diseases onAe. speltoideschromosome 2S, and the resistance genes for FHB disease onTh. elongatumchromosome 7E.We have evaluated multiple homozygous recombinant lines involving critical regions of the chromosomes to map the resistance genes and to select the recombinants with shortest alien segments for germplasm development.In addition, we have been minimizing theAe. speltoidesandTh. elongatumsegments containing the genes of interest to reduce potential linkage drag by inducing additional homoeologous recombination.Also, we have been incorporating the wild species-derived resistance genes into adapted US wheat genotypes for breeding-ready germplasm development using marker-assisted backcrossing breeding scheme.We will continue developing homozygous recombinant lines involving other chromosomes and evaluating the traits targeted in this project for gene identification and mapping.The newly-identified genes will be introgressed into adapted wheat backgrounds for germplasm development.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Zhang, W., Zhu, X., Zhang, M., Shi, G., Liu, Z., and Cai, X. 2019. Chromosome engineering-mediated introgression and molecular mapping of novel Aegilops speltoides-derived resistance genes for tan spot and Septoria nodorum blotch diseases in wheat. Theor Appl Genet 132: 26052614.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Gyawali, Y., Zhang, W., Chao, S., Xu, S. S., and Cai, X. 2019. Delimitation of wheat ph1b deletion and development of ph1b-specific DNA markers. Theor Appl Genet 132:195204.
  • Type: Book Chapters Status: Published Year Published: 2019 Citation: Zhang, W., and Cai, X. 2019. Alien Introgression and breeding of synthetic wheat. In: Advances in Breeding Techniques for Cereal Crops. Ed by Frank Ordon and Wolfgang Friedt. Burleigh Dodds Science Publishing, pp3-30.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhang, M., Zhang, W., Zhu, X., Sun, Q., Chao, S., Yan, C., Xu, S. S., Fiedler, J., and Cai, X. 2020. Partitioning and physical mapping of wheat chromosome 3B and its homoeologue 3E in Thinopyrum elongatum by inducing homoeologous recombination. Theor Appl Genet 133:12771289.
  • Type: Journal Articles Status: Submitted Year Published: 2020 Citation: Zhang, M., Zhang, W., Zhu, X., Sun, Q., Yan, C., Xu, S. S., Fiedler, J., and Cai, X. 2020. Dissection and physical mapping of wheat chromosome 7B by inducing meiotic recombination with its homoeologues in Aegilops speltoides and Thinopyrum elongatum. Theor Appl Genet
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Morris, C. F., Ibba, M. I., Zhang, M., and Cai, X. 2019. Identification of a conserved ph1b-mediated 5DS-5BS crossing over site in soft-kernel durum wheat (Triticum turgidum subsp. durum) lines. Euphytica 215: 200.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Morris, C. F., Kiszonas, A. M., Murray, J., Boehm Jr., J., Ibba, M. I., Zhang, M., and Cai, X. 2019. Re-evolution of durum wheat by introducing the hardness and Glu-D1 loci. Frontiers in Sustainable Food Systems 3 (103).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Cai, X., Zhang, W., Zhang, M., Gyawali, Y., Zhu, X., Cao, Y., Naraghi, S. M., Ren, S., Long, Y., Shi, G., Zhang, Q., Sun, Q., Ma, G., Liu, Z., Yan, C., Chao, S., and Xu, S. S. 2019. Diversification and understanding of the wheat B genome by homoeologous recombination and comparative genome analysis (poster). Proc. 1st Intern. Wheat Congress, Saskatoon, Canada. July 21-26, 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Cai, X. 2019. Genomics-enabled chromosome engineering for alien introgression and genome characterization in wheat (invited talk). The Joint 2019 National Association of Plant Breeders Meeting and NIFA-AFRI PD Meeting, August 25-29, 2019, Georgia.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Zhang, W., Zhu, X., Zhang, M., Shi, G., Liu, Z., and Cai, X. 2019. Chromosome engineering-mediated molecular mapping and introgression of novel Aegilops speltoides-derived resistance genes for tan spot and Septoria nodorum blotch diseases in wheat (poster). Plant & Animal Genome XXVII, San Diego, CA, January 12-16, 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Ren, S., Zhu, X., Leng, Y., Zhang, W., Talukder, Z., Zhong, S., Fiedler, J., Qi, L., and Cai, X. 2019. Molecular mapping of hexaploid wheat-derived Fusarium head blight resistance in durum wheat (poster). 2019 National Fusarium Head Blight Forum, Milwaukee, Wisconsin. December 8-10, 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Ibba, M. I., Boehm Jr., J. D., Kiszonas, A. M., Zhang, M., Cai, X., and Morris, C. F. 2019. Identi?cation of a conserved ph1b-mediated 5DS-5BS crossing over site in soft-kernel durum wheat (Triticum turgidum subsp. durum) lines (poster). Proc. 1st Intern. Wheat Congress, Saskatoon, Canada. July 21-26, 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Long, Y., Sun, Q., Cai, X., Faris, J. D., Harris, M. O., Wulff, B. B. H., Lagudah, E., and Xu, S. S. 2019. Nested loop PCR (NL-PCR) for amplification of large DNA fragments with complex structure in wheat (poster). Proc. 1st Intern. Wheat Congress, Saskatoon, Canada. July 21-26, 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Zhang, Q., Long, Y., Friesen, T. L., Jin, Y., Rouse, M. N., Cai, X., Dykes, L., Xu, S. S. 2019. Elimination of yellow pigment gene tightly linked to stem rust resistance gene Sr43 derived from Thinopyrum ponticum (poster). Proc. 1st Intern. Wheat Congress, Saskatoon, Canada. July 21-26, 2019.
  • Type: Theses/Dissertations Status: Published Year Published: 2020 Citation: Zhang, M. 2020. Homoeologous recombination-based chromosome engineering for physical mapping and introgression in wheat and its relatives Aegilops speltoides and Thinopyrum elongatum. North Dakota State University, Fargo, ND