Source: MICHIGAN STATE UNIV submitted to NRP
BTT EAGER: BREAKING THE WHEAT DOMESTICATION BOTTLENECK BY INTERPLOIDY HYBRIDIZATION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1018278
Grant No.
2019-67013-29015
Cumulative Award Amt.
$300,000.00
Proposal No.
2018-09047
Multistate No.
(N/A)
Project Start Date
Dec 15, 2018
Project End Date
Dec 14, 2021
Grant Year
2019
Program Code
[A5173]- Early Concept Grants for Exploratory Research (EAGERs) to Develop Breakthrough Ideas and Enabling Technologies to Advance Crop Breeding and Functional Genomics
Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824
Performing Department
PLANT SOIL MICROBIAL
Non Technical Summary
Manipulation of genes imprinted in grain endosperm will enable hybridizations between hexaploid wheat, Triticum aestivum with wild diploid relatives. Hexaploid wheat mutants have been identified with nonsense or missense mutations in all three wheat homoeoalleles of the Arabidopsis maternally expressed Polycomb Repressive Complex FIS2-PRC2 genes that establish endosperm imprinting: FIE, FIS2, MEA as well as the paternally expressed genes PEG2 and PEG9. Mutations in each of these genes have been demonstrated to restore endosperm functionality in crosses between different ploidy levels. The wheat orthologues will be pyramided in all possible combinations of A, B and D genome mutations. All combinations of wild type, single, double and triple mutant T. aestivum genotypes will be crossed as females to diploid Ae. tauschii and T. monococcum genotypes. It will be determined which FIS-PRC2 mutations restore endosperm and compatibility to interploidy crosses.Gene expression will be evaluated in developing endosperm tissue from crosses with mutant and euploid wheat. Whole genome sequence will be generated for the Cadenza parent and one Ae. tauschii and T. monococcum accession. Gene expression profiles will be generated using RNA sequencing (RNASeq). Expression will be investigated for normal 6x and 2x hybridizations as well as successful and unsuccessful inter-ploidy hybridizations. A whole transcriptome approach will elucidate the role of FISPRC2 mutations on all genes and pathways orchestrated during endosperm development. Further, expression data will provide a genetic mechanism for failure and restoration of endosperm in crosses between ploidy levels.The FIS2-PRC complex is highly conserved across all eukaryotic organisms. Fundamental work in Arabidopsis has identified the genes and their functions in plants. Orthologues in rice have been experimentally verified to have similar functions. This work will determine the pattern of allele-specific expression of FIS2-PRC genes in both diploid and polyploid wheat species. The endosperm functions as a barrier to gene flow between related species of different ploidy levels. Through this work, gene flow will be restored between species separated by millennia of natural and artificial selection. This work will determine the role of FIS-PRC2 genes as a hybridization barrier between species.Restoring direct hybridization between hexaploid and diploid wheat will open the entire gene pool of diploid wheat relatives for wheat improvement. Hexaploid wheat has been under selection for agronomic performance and adaptation to modern farming practices. In wild relatives, selection over millennia has favored alleles conferring adaptation these harsh environments. Through this work, the genes conferring resistance to disease and environmental extremes can be transferred directly to any hexaploid wheat background. The entire breadth of diploid wheat genetic resources may be used to improve hexaploid wheat.Many single locus disease resistance genes have been transferred from Ae. tauschii and T. monococcum to wheat which requires only transfer of single chromosome segments. However, to access quantitative variation for traits like grain yield, water use efficiency, high temperature stress tolerance requires the entire genome of an accession must be transferred. Through this work, populations may be constructed to capture the breadth of valuable quantitative traits in wild diploid wheat relatives.Wheat consumption very closely tracks wheat production and several successive years of greater than 5% underproduction of wheat can lead to a rapid depletion of global wheat stocks. Access to genetic variation to improve hexaploid wheat will support a sustainable supply of grain to a growing population. The most populated areas on earth rely on wheat as a staple food source. It is the most highly populated areas that experience increasingly extreme wheat production conditions. The same genes that promote survival of wild relatives in extreme environments will allow hexaploid wheat to survive under extreme production conditions.
Animal Health Component
25%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011549108050%
2061549105025%
2021549108025%
Knowledge Area
206 - Basic Plant Biology; 202 - Plant Genetic Resources; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1549 - Wheat, general/other;

Field Of Science
1080 - Genetics; 1050 - Developmental biology;
Goals / Objectives
A. Enable fertile direct hybridizations between hexaploid wheat and diploid relatives using FIS-PRC2 mutants.Restoring endosperm formation in direct hybrids between hexaploid and diploid wheat can be achieved by two mechanisms. First, autonomous endosperm may be produced in wheat plants carrying mutations in all three homoeoalleles of FIE, FIS2 or MEA. Wheat FIS-PRC2 mutants are expected to produce functional endosperm in crosses with diploids. Second, introducing mutations in the maternally expressed genes will counter the maternal dominance of the hexaploid female and restore the 2:1 balanced maternal:paternal allele specific expression. In the target mutant genotypes, the expression of FIS-PRC2 genes is expected to be reduced to that of a lower ploidy level. For example, the genotype fie-7A/ fie-7B/FIE-7D is expected to have diploid levels of expression. This reduction in the expression of maternal FIE alleles will restore the 2:1 ratio of maternal to paternal alleles in the endosperm.B. Evaluate the expression of imprinted FIS-PRC2 genes in mutant and wild type direct hybrids.The RNA-Seq experiments proposed in this work will elucidate the expression levels and parent of origin for the maternally and paternally expressed genes during compatible and incompatible hybridizations within and across ploidy levels. In this objective, the expression level of all FIS-PRC2 genes will be evaluated. Comparisons of A, B and D genome homoeoalleles can be made to determine which allele functions primarily in endosperm formation.
Project Methods
A. Hybridizing hexaploid FIS-PRC2 mutants with diploid Ae. tauschii 1. MEG and PEG mutants. Mutants of the hexaploid wheat variety 'Cadenza' (http://www.wheat-tilling.com) have been identified with nonsense or missense mutations in all three wheat homoeoalleles of the FIS-PRC2 components FIE, FIS2, and MEA (Table 1). Wheat orthologues of PEGs involved in restoration of endosperm development have been identified including PEG2-1A, PEG2-1D and PEG9-3A. Crosses have been initiated to pyramid all combinations of FIS-PRC2 mutations and PEGs in the A, B and D genomes. For each FIS-PRC2 gene, the target genotypes will be homozygous for mutant alleles at one, two or all three homoeoalleles of the A, B and D genomes. All mutants undergo normal vegetative and reproductive development. Most importantly, all MEG and PEG mutants produce viable female and male gametophytes, respectively.2. Interploidy hybridizations. FIS-PRC2 mutants of hexaploid wheat will be hybridized directly with diploid Ae. tauschii including the Ae. tauschii reference genome accession AL8/78 (Luo et al., 2017). Additional accessions will include TA2477, TA1691, CIae23, TA1715 and TA2478 which demonstrate high levels of type II resistance to Fusarium head blight (Brisco et al., 2017). All combinations of wild type, double and triple mutant hexaploid genotypes will be crossed with diploid Ae. tauschii accessions. MEG mutants will be used as females in crosses with diploids as males. PEG mutants will be used as males and diploids will be used as females.Seed from compatible crosses that produce viable endosperm will be germinated in the lab using a standard cold imbibition of five days followed by germination at ambient temperatures. A viable seed from a compatible hybridization will be determined as a seed that germinates, produces of seedlings which undergo normal vegetative growth followed by reproductive development and flowering. Viable plants will be backcrossed to the 'Cadenza' recurrent parent to generate BC1F1 plants.3. Endosperm microscopy. The endosperm composition of crosses with FIS-PRC2 and PEG mutants will be investigated to provide insight into the mechanism of endosperm restoration. Tissue sections will be taken at 7, 12, 14 and 21 days after pollination to visualize endosperm development in compatible and incompatible crosses. Observations will be made of embryo and endosperm development to determine the point at which endosperm failure takes place during incompatible hybridizations. The endosperm constitution of compatible and incompatible hybridizations will be compared to normal endosperm to determine the differences in morphology of endosperm cells. 4. Chromosome counts. Metaphase chromosomes will be counted in root tips of 6X/2X and 2X/6X hybrids to confirm the expected n=28 chromosomes (21 T. asestivum, 7 Ae. tauschii). Chromosome counts will be made in the BC1F1 to determine the range of aneuploidy due to random segregation of A and B genome chromosomes.B. Evaluating gene expression during compatible and incompatible hybridizations1. Targeting cross combinations for gene expression studies. A total of 36 cross combinations will be targeted for RNA-Seq. First, hybridizations between all wheat FIS-PRC2 mutants and diploid wild relatives will determine the compatible and incompatible crosses. In hybridizations between hexaploid wheat and Ae. tauschii, endosperm failure occurs between 13 and 18 days after pollination (Olson, unpublished). Therefore, RNA will be sampled at 14 days after pollination for both compatible and incompatible hybridizations. This time point is optimum for detecting the greatest differential expression genes in compatible and incompatible interactions.2. RNA isolation from interploidy hybrids. RNA will be isolated from whole ground caryopses from three biological replicates of each cross using the Qiagen RNeasy PowerPlant Kit (Cat No: 13500-50), treated with DNAase and Tru-Seq Illumina Stranded RNA-Seq libraries constructed. Libraries will be pooled and sequenced on an Illumina HiSeq4000 at the MSU Research Technology Support Facility (RTSF; 50 nucleotide, single end) generating a minimum of 50M paired-end reads per sample. RNA-Seq reads will be evaluated for quality using FASTQC (www.bioinformatics.babraham.ac.uk/projects/fastqc), cleaned for quality and adaptors using CutAdapt (Martin, 2017), and aligned to the Chinese Spring hexaploid reference genome (https://www.wheatgenome.org/Projects/IWGSC-Bread-Wheat-Projects/Reference-genome/IWGSC-Reference-Wheat-Genome) using TopHat2 (Trapnell et al., 2009). Normalized gene expression levels will be calculated using Cufflinks (Trapnell et al., 2010) and reported as fragments per kilobase of exon model per million fragments mapped (FPKM).3. DNA isolation from parental lines and determination of imprinted genes. A 5X whole genome sequence (WGS) will be developed for the 'Cadenza' wheat parent and the Ae. tauschii accession TA2477. High molecular weight DNA will be isolated from young etiolated leaf tissue of the parents using a modified CTAB method and Illumina Tru-Seq Nano genomic DNA libraries constructed and sequenced on the HiSeq4000 (150 nt paired-end reads) generating 5X coverage of each genome. WGS reads will be evaluated for quality using FASTQC (www.bioinformatics.babraham.ac.uk/projects/fastqc), cleaned for quality and adaptors using CutAdapt (Martin, 2017), and aligned to the Chinese Spring hexaploid reference genome (IWGSC RefSeq v1.0) using BWA-MEM (Li, 2013). PCR duplicates will be removed, and high quality sequence variants called as described previously (Pham et al., 2017). To determine levels of imprinted genes in parental and hybrid endosperm, RNA-Seq reads will be filtered for uniquely mapping and high quality alignments and read depths at biallelic positions counted as previously described (Pham et al., 2017). Imprinted genes will be defined as described previously (Yang et al., 2018).C. Crossing FIS-PRC2 mutations into elite hexaploid wheat varieties1. Backcrossing of FIS-PRC2 mutations. A set of spring and winter wheat varieties will be crossed with FIS-PRC2 mutants to initiate transfer of the mutant alleles into elite regional wheat backgrounds. Spring wheat genotypes will include 'Seahawk' and 'Elgin'. Winter wheat genotypes will include 'Japser', 'Hilliard', 'Overland' and 'Zenda'. A total of three backcrosses will be made with large populations of at least 300 backcross seed each generation. The BC3F1 will be self-pollinated and fixed mutants will be identified in the BC3F2. The 'Cadenza' donor background is a spring type and backcrosses will be made using spring types to avoid vernalization. For winter wheat backgrounds, winter types will be selected at the BC3F2.

Progress 12/15/18 to 12/14/21

Outputs
Target Audience:Results of the project have been communicated to the plant breeding community internally to MSU colleagues at a seminar and locally in lab meetings to our research group. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate student, Selena Lopez, received her MS degree from Michigan State University. Selena is now a PhD student in wheat breeding and genetics at Colorado State University. Undergradate researcher Samantha Mitchell received training in plant sciences through this project. How have the results been disseminated to communities of interest?Results have been communicated in writing through a MS thesis by Selena Lopez and a seminar to the MSU plant sciences community. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? A set of 14 FIE, 7 FIS2 and 22 MEA mutants were hybridized directly with Aegilops tauschii. AD mutants for FIS2 facilitated the formation of near wild type levels of caryopses in direct hybridizations. Double AD and BD mutants as well as triple MEA mutants facilitated direct hybridization to near wild type levels as well. Double mutants of FIE did not facilitate direct hybridization. Triple mutants of FIE could not be recovered. Table was requested by C. Tobias to be removed as the table did not upload properly. Note that the table was clarifying that sequencing was intended for genotyping purposes. As well Objective B could not be completed in the grant timeframe due to COVID restrictions.

Publications


    Progress 12/15/19 to 12/14/20

    Outputs
    Target Audience:The plant breeding community has been reached through a poster presentation at a well attended symposium in February, 2020. COVID-19 prevented the dissemination of results through oral presentations. No additional oral or poster presentations could be made in 2021. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has provided excellent training for MS student, Selena Lopez. Selena has developed an excellent background in molecular genetics, classical genetics and bioinformatics. The training made possible in this work is providing the background necessary for an impactful career in plant genetics. Selena has learned excellent project management skills in this project. The highly complex crossing schemes requires diligence in record keeping and organization. Selena has developed communication skills through presenting her work to the plant breeding community through a poster presentation as well as internally to MSU colleagues and locally in lab meetings to our research group. Selena is now a PhD student in wheat breeding and genetics at Colorado State University with Dr. Esten Mason. How have the results been disseminated to communities of interest?COVID-19 prevented travel to present on this project. What do you plan to do during the next reporting period to accomplish the goals?Results from direct hybridization of target mutant genotyps with Aegilops tauschii will be presented in the Final Report. The gene expression study will be completed beyond the life of this project.

    Impacts
    What was accomplished under these goals? Cycle 4 Progeny Progeny from Cycle 3 were genotyped via amplicon sequencing. Genotyping screened for homozygous Cadenza triple mutant lines (aabbdd) and homozygous Cadenza double mutant lines (aaB_dd, A_bbdd, and aabbD_). Cycle 3 progeny lines were expected to occur at 6.25% or 3.125% homozygous triple mutants (aabbdd). Amplicon sequencing Cycle 3 A subset of lines that were expected to be double or triple homozygous mutant from KASP were genotyped using amplicon sequencing. A total of 20 Cycle 3 lines were genotyped, along with the mutant donor parents and wild Cadenza. Cycle 2 progeny were expected to occur at 12.5% for heterozygous mutations in three homeologs (AaBbDd), and 6.25% for heterozygous mutations in two homeologs and a homozygous mutation in one homeolog (AaBbdd, aaBbDd, or AabbDd). Amplicon sequencing confirmed MEA genotypes and elucidated further information for FIS2 and FIE samples (Table 2.10). The target genotype (AaBbDd, AaBbdd, aaBbDd, or AabbDd) could not be obtained for FIS2. Genotype MEA FIE FIS2 AaBbDd 3 1 0 AabbDd 1 0 0 AaBbdd 1 0 0 AaBbDD 0 7 0 AaBBDd 0 0 1 AaBBDD 0 7 1 AABBDD 0 0 3 total 5 15 5 Table 2.10: Cycle 3 amplicon sequencing genotypic distribution Amplicon sequencing Cycle 4 A total of 509 Cycle 4 lines were genotyped, along with the mutant donor parents and wild Cadenza (Table 2.11). Desired Cycle 3 progeny were expected to occur at 3.125% if the parental genotype was heterozygous for all three homeologs (AaBbDd) or 6.25% if the parental genotype had heterozygous mutations in two homeologs and a homozygous mutation in one homeolog (AaBbdd, aaBbDd, or AabbDd). FIE samples sequenced had parental genotypes of the former (AaBbDd) while MEA samples sequenced had parental genotypes of the latter (AaBbdd and AabbDd). Even though there were no homozygous triple mutant FIE lines, this was within the range of the expected distribution (P>0.05). MEA homozygous triple mutants occurred at a slightly lower rate than expected, but only slightly at P=0.047. (Table 2.12). Homozygous triple mutant FIS2 lines were unable to be obtained. MEA FIE FIS2 aabbdd 2 0 0 AaBbDd 0 1 0 AabbDd 10 1 0 aaBbDd 0 5 0 aabbDd 9 0 0 aaBBdd 0 0 0 aaBBDd 0 0 1 AaBBDd 0 1 7 AaBBdd 1 0 0 AAbbdd 0 0 0 AABbDd 0 3 0 AABbdd 0 0 0 AAbbDd 6 0 0 aabbDD 16 0 0 AaBbDD 9 94 0 AabbDD 23 24 0 aaBbDD 5 46 0 AaBBDD 8 15 62 aaBBDD 3 6 17 AABbDD 7 58 0 Table 2.11: Cycle 4 genotyping results. MEA FIE FIS2 AAbbDD 10 18 0 AABBDd 0 0 3 AABBdd 0 0 0 wild 5 7 26 total 114 279 116 Table 2.11 (cont'd): Cycle 4 genotyping results. Genotype Observed occurrence Expected occurrence chi square value P-value MEA aabbdd 2 7.125 3.93 0.047 FIE aabbdd 0 1.656 1.709 0.191 Table 2.12: Cycle 4 chi square results

    Publications


      Progress 12/15/18 to 12/14/19

      Outputs
      Target Audience:The plant breeding community has been reached through a poster presentation at a well attended symposium in February, 2020. Changes/Problems:Funding for the project became available in December, 2018. A MS student, Selena Lopez, began work in June, 2019. For six months, the project PI carried out project objectives. After the student began work, it was determined that genotyping errors had occurred in identifying mutant parents for the initial F1 crosses made by the PI. Selena genotyped new mutant stocks, made the F1s and the F1 intercrosses to get the project back on track. The genotyping error cost the project six months. The delay has pushed back the timeline of the gene expression studies. An extension is being requested to enable the gene expression studies. Further, COVID-19 resulted in a halt of lab activites from March 2020 through June 2020. During this time, no work could be completed in the lab. Plants could only be grown in the greenhouse keeping the generations advancing. Sequencing facilities at MSU and across the US were closed and genotyping could not be complted until August, 2020. COVID-19 caused signifiant delays in genotyping and identifying target genotypes slowing progress towards project goals. What opportunities for training and professional development has the project provided?The project has provided excellent training for MS student, Selena Lopez. Selena has developed an excellent background in molecular genetics, classical genetics and bioinformatics. The training made possible in this work is providing the background necessary for an impactful career in plant genetics. Selena has learned excellent project management skills in this project. The highly complex crossing schemes requires diligence in record keeping and organization. Selena has developed communication skills through presenting her work to the plant breeding community through a poster presentation as well as internally to MSU colleagues and locally in lab meetings to our research group. How have the results been disseminated to communities of interest?Results of the project have been communicated to the plant breeding community through a poster presentation as well as internally to MSU colleagues and locally in lab meetings to our research group. What do you plan to do during the next reporting period to accomplish the goals?The target PRC2 genotypes will be identified and crossed directly with diploid Aegilops tauschii. Gene expression studies will be carried out. PRC2 mutant alleles will be transferred to elite hard and soft winter wheat and spring wheat genotypes paving the way for large scale introgression of Aegilops tauschii allelles into wheat.

      Impacts
      What was accomplished under these goals? A. Enable fertile direct hybridizations between hexaploid wheat and diploid relatives using FIS-PRC2 mutants. Seed of PRC2 mutants were genotyped to identify plants homozygous for mutations in MEA, FIS2 and FIE alleles. Mutants were intercrossed in pairwise combinations, A'/B', A'/B', B'/D' (' indicates a mutant allele). A total of 17 crosses were made to generate F1s between A, B and D mutant alleles for MEG and PEG genes. A total of 91 F1 seed were generated and grown for intercrossing. Crosses were then made among the mutant F1s to generate intercross F1s homozygous for at least one mutant allele and heterozygous for two mutant alleles. For example, one target genotype was A'A'BB'DD' homozygous for mutant A genome alleles and heterozygous for B and D genome alleles. A total of 17 successful unique intercrosses were made. From these crosses, 73 intercross F1 seed were planted and genotyped to identify plants for a subsequent round of intercrossing. Plants heterozygous for two mutant alleles were identified among the intercross F1 plants and were intercrossed. Crosses were made between plants heterozygous for one common mutant allele and two contrasting mutant alleles. For example the AA'BB'DD genotype was crossed with the AABB'DD' genotype. The target progeny genotype for this cross is AA'B'B'DD', homozygous for one mutant allele and heterozygous for two mutant alleles. The F1 progenies from the second intercross were genotyped by amplicon sequencing of PRC2 targets. Currently, the F2 progeny of target genotypes homozygous for two and three mutant alleles are being identified. The target genotypes will be used to test the hypothesis regarding direct hybridization. B. Evaluate the expression of imprinted FIS-PRC2 genes in mutant and wild type direct hybrids. Work on this objective will take place once the target mutant genotypes have been identified.

      Publications

      • Type: Other Status: Published Year Published: 2020 Citation: S. Lopez, E.Olson. Bypassing endosperm failure in interspecific crosses of Triticum aestivum (Wheat) . Poster at the 5th Biennial Symposium on Plant Breeding at North Carolina State University, February 5th, 2020.