Progress 10/01/16 to 09/30/21
Outputs
Target Audience:We have two targets: plant breeders, and plant chromosome biologists. Our communications have been with sponsoring (and potentially sponsoring) seed companies (Rjik Zwaan, East-West Seeds, Syngenta, Sakata Seed America) and with other labs here at UC Davis. We have given presentations on our work for both seed companies (largely at Davis) and research scientists (at research symposia). Some of our work has been published (listed below). Changes/Problems:As described above, we came up with a nontransgenic approach to create novel CENH3 alleles and employed this in rice and tomato. However, tomato is more sensitive to the effects of mutations in CENH3, in terms of its development and fertility, and less sensitive than arabidopsis to effects on haploid induction. What opportunities for training and professional development has the project provided?Undergraduate and postdocs have been trained in plant genetics and molecular biology. How have the results been disseminated to communities of interest?See above. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
To review our goals: Arabidopsis plants carrying mutations in the gene encoding CENH3, when outcrossed by pollen from plants wild-type for CENH3, can produce uniparental haploids derived only from the wild-type pollen's genome. In order to determine whether this effect can be reproduced in crop plants, we will create lines of tomato that have lost the wild-type CENH3 allele and carry instead a modified CENH3. This goal can be achieved two ways: 1) Elimination or modification of the wild-type allele via CRISPR technology, accompanied by complementation of (essential) CENH3 function by a mutant allele. This mutant allele might be introduced transgenically, in a plant defective for the endogenous allele. Originally, we planned to complement the CENH3 knockout allele with two very different classes of transgenic CENH3 alleles--alleles encoding subtle, single amino acid changes shown to induce haploids at approximately 15% of progeny in Arabidopsis, and a more elaborate, chimeric, trans-kingdom fusion protein that produces haploids in the majority of Arabidopsis progeny. 2) However, during the course of this funding, we found the endogenous locus can be modified (by CRISPR, a site-specific gene editor) to create "weak" alleles. Null alleles (complete loss of function) are commonly induced by CRISPR, but in-frame small inserts or deletions at the CRISPR target site are also found. So this provided another avenue for the create of crop plants carrying subtle mutations in CENH3, which might be haploid-inducers. These plants can then be tested for their frequency of haploid induction. Progress: Since the initiation of this project in 2016, we have discovered that, in Arabidopsis, CRISPR mutagenesis can be employed to create haploid-inducing alleles of CENH3 at the native locus, by inducing in-frame deletions or insertions (indels) at the endogenous locus. These haploid-inducing (HI) plants are gene-edited, not transgenic. The Arabidopsis mutants grow well and are fully fertile, but at the same time can be excellent HI parents, as females (see Kuppu et al 2020 journal article). We are replicating these studies in tomato, and have isolated many viable in-frame mutations in CENH3. We have been investigating the effects of these alleles on growth, fertility and haploid induction ad have largely abandoned our earlier approach of introducing transgenes into knockout (KO) lines. Our CRISPR approach has also generated many KO alleles which may be useful for such studies in the future, but it is not clear that null alleles are transmissible in tomato. The transmissibility (in haploid pollen and ova) of each CENH3 may depend on both on its own ability to express functional the "quality" of the and the ability of persisting mRNA and protein from the homolog in the maternal tissue to support the defective haploid gametophyte. allele on the homolog. Our preliminary results suggest that tomato is far more sensitive to the effects of small in-frame insertions and deletions on growth, development, and fertility- effects not observed at all using similar or identical alleles in Arabidopsis. Our current hypothesis is that for CENH3-based HI to work in tomato it will have to be "tuned" such that the CENH3 defect is subtle enough that the plant remains fertile yet defective enough that HI can be induced. We did perform an extensive assay for induction of haploids by a tomato line homozygous for an in-frame, 6 nt deletion in CENH3- identical to a mutation in Arabidopsis that is haploid inducing, using the mutant plant as a female and a wild-type plant as a pollen source. The male parent carried a recessive mutation, affecting plant color that would allow us to easily observe paternal haploids. Among 600 progeny, however, no haploids were observed. it is possible that the differences in reproductive biology between tomato and Arabidopsis are such that this approach may not work in tomato. It may work in more "Arabidopsis-like" crops. Future directions: A recent publication on CENH3-based haploid induction in maize indicated that heterozygotes for knockouts in CENH3 can act as haploid inducers when outcrossed by wild-type pollen. We have employed CRISPR to generate heritable mutations in CENH3 in rice, and look forward to characterizing these plants for HI.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
A variety of changes, including CRISPR/Cas9-mediated deletions, in CENH3 lead to haploid induction on outcrossing
Sundaram Kuppu 1, Mily Ron 1, Mohan P A Marimuthu 1 2, Glenda Li 1, Amy Huddleson 1, Mohamed Hisham Siddeek 1, Joshua Terry 1, Ryan Buchner 1, Nitzan Shabek 1, Luca Comai 1 2, Anne B Britt Plant Biotechnol J 2020 Feb 24;18(10):2068-2080.
doi: 10.1111/pbi.13365.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Epigenetically mismatched parental centromeres trigger genome elimination in hybrids
MOHAN P. A. MARIMUTHU, RAVI MARUTHACHALAM, RAMESH BONDADA,SUNDARAM KUPPU, EK HAN TAN, ANNE BRITT, SIMON W. L. CHAN, AND LUCA COMAI
SCIENCE ADVANCES " 19 Nov 2021 " Vol 7, Issue 47 " DOI: 10.1126/sciadv.abk1151
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2018
Citation:
Prospects for CENH3-based haploid induction, A. B. Britt, XXVI International Eucarpia Symposium Section Ornamentals: Editing Novelty 2018, Erfurt, Germany
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2018
Citation:
A variety of subtle changes in CENH3 result in haploid induction. Anne Britt, Mily Ron. Sundaram Kuppu, EMBO Workshop Plant Genome Stability and Change 2018, Erfurt, Germany
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Progress 10/01/19 to 09/30/20
Outputs
Target Audience:We have two targets: plant breeders, and plant chromosome biologists. Our communications have been with sponsoring (and potentially sponsoring) seed companies (Rjik Zwaan, East-West Seeds, Syngenta, Sakata Seed America) and with other labs here at UC Davis. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Undergraduates were trained in genetics and molecular genetics. How have the results been disseminated to communities of interest?Some of the results have been described in the Kuppu et al 2020 reference described above. Others have been described in private reports to our sponsors. What do you plan to do during the next reporting period to accomplish the goals?A good candidate allele in tomato (a 6 nt deletion) has been isolated and homozygous, nontransgenic plants derived. These are currently being characterized for rate of HI and effects on genomic stability. A wider range of CRISPR-induced mutations at the CENH3 locus of Arabidopsis are being characterized for their effects on HI.
Impacts
What was accomplished under these goals?
To review our goals: Arabidopsis plants carrying mutations in the gene encoding CENH3, when outcrossed by pollen from plants wild-type for CENH3, can produce uniparental haploids derived only from the wild-type pollen's genome. In order to determine whether this effect can be reproduced in crop plants, we will create lines of tomato that have lost the wild-type CENH3 allele and carry instead a modified CENH3. This requires: A) Elimination or modification of the wild-type allele via CRISPR technology B) Complementation of (essential) CENH3 function by a mutant allele. This mutant allele might be introduced transgenically, in a plant defective for the endogenous allele, or might be a mutation at the endogenous CENH3 locus. We planned to complement the CENH3 knockout allele with two very different classes of transgenic CENH3 alleles--Alleles encoding subtle, single amino acid changes shown to induce haploids at approximately 15% of progeny in Arabidopsis --A more elaborate, chimeric, trans-kingdom fusion protein that produces haploids in the majority of Arabidopsis progeny. C) Alternatively, the endogenous locus can be modified (by CRISPR) to create "weak" alleles. Null alleles (complete loss of function) are commonly induced by CRISPR, but in-frame small inserts or eletions at the CRISPR target site are also found. D) These plants will then tested for their frequency of haploid induction. Progress: Since the initiation of this project in 2016, we have discovered that, in Arabidopsis, CRISPR mutagenesis can be employed to create haploid-inducing alleles of CENH3 at the native locus, by inducing in-frame deletions or insertions (indels) at the endogenous locus. These haploid-inducing (HI) plants are gene-edited, not transgenic. The Arabidopsis mutants grow well and are fully fertile, but at the same time can be excellent HI parents, as females (see Kuppu et al 2020 journal article cited above). We are replicating these studies in tomato, and have isolated many viable in-frame mutations in CENH3. We have been investigating the effects of these alleles on growth, fertility and haploid induction ad have largely abandoned our earlier approach of introducing transgenes into knockout (KO) lines. Our CRISPR approach has also generated many KO alleles which may be useful for such studies in the future, but it is not clear that null alleles are transmissible in tomato. Their transmissibility may depend on the "quality" of the allele on the homolog. Our preliminary results suggest that tomato is far more sensitive to the effects of small in-frame indels on growth, development, and fertility- effects not observed at all using similar or identical alleles in Arabidopsis. Our current hypothesis is that for CENH3-based HI to work in tomato it will have to be "tuned" such that the CENH3 defect is subtle enough that the plant remains fertile yet defective enough that HI can be induced. However, it is possible that the differences in reproductive biology between tomato and Arabidopsis are such that this approach may not work in tomato though it may work in more "Arabidopsis-like" crops.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
A variety of changes, including CRISPR/Cas9?mediated deletions, in CENH3 lead to haploid induction on outcrossing
Sundaram Kuppu Mily Ron Mohan P.A. Marimuthu Glenda Li Amy Huddleson Mohamed Hisham Siddeek Joshua Terry Ryan Buchner Nitzan Shabek Luca Comai Anne B. Britt &
First published: 24 February 2020 https://doi.org/10.1111/pbi.13365
|
Progress 10/01/18 to 09/30/19
Outputs
Target Audience:We have two targets: plant breeders, and plant chromosome biologists. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Several undergraduates and junior researchers have been trained in molecular genetics and plant genetics as part of this project. How have the results been disseminated to communities of interest?This project was presented at an international meeting focused on horticultural breeding (Eucarpia 2019, Section Ornamentals, Erfurt, Germany). It was also discussed with several seed companies (Rjik Zwaan, East-West Seeds, Syngenta, BASF), and at Seed Central events here at UC Davis. We also have a paper in revision for Plant Biotechnology. What do you plan to do during the next reporting period to accomplish the goals?We will focus on determining whether the null allele is transmissible in a wt/null heterozygote, and on the analysis of aneuploidy in outcrossed progeny.
Impacts
What was accomplished under these goals?
To review our goals: Arabidopsis plants carrying mutations in the gene encoding CENH3, when outcrossed by pollen from plants wild-type for CENH3, can produce uniparental haploids derived only from the wild-type pollen's genome. In order to determine whether this effect can be reproduced in crop plants, we will create lines of tomato and potato that have lost the wild-type CENH3 allele and carry instead a modified CENH3. This requires: A) Elimination or modification of the wild-type allele via CRISPR technology B) Complementation of (essential) CENH3 function by a mutant allele. This mutant allele might be introduced transgenically, or might be a mutation at the endogenous CENH3 locus. We will complement the CENH3 knockout allele with two very different classes of CENH3 mutants: --Alleles encoding subtle, single amino acid changes shown to induce haploids at approximately 15% of progeny in Arabidopsis --A more elaborate, chimeric, trans-kingdom fusion protein that produces haploids in the majority of Arabidopsis progeny. C) These plants will then tested for their frequency of haploid induction. During the last year, our potato work has been handed over to our collaborators in the Comai lab- we are no longer involved with this crop. We have employed CRISPR/cas9 to generate several CRISPR mutant lines of tomato, as well as tomato lines carrying a chimeric construct mimicking the Arabidopsis "GFP-tailswap" haploid inducer as a transgene. Unlike the effects seen in Arabidopsis, this transgene does not rescue the lethal phenotype of a cenh3 null mutant in tomato. It does, however, improve the growth and fertility of weak, semifunctional alleles. Our preliminary data also suggests that a null allele, in a heterozygous plant, cannot be transmitted- also unlike the result in Arabidopsis. However, we will further investigate this effect in the coming year. We have developed lines of tomato that carry in-frame small deletions of CENH3 at the native locus. These lines are essentially an allelic series, with increasing deleterious effects on growth and fertility depending on the location and extent of the deletion. We have discovered that, while similar- even identical- deletions do induce haploids in Arabidopsis, our "delta six" deletion in the beginning of the histone fold domain, while viable and fertile, does not induce haploid when outcrossed by wild-type pollen. Aneuploid progeny have been observed, however.
Publications
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:Our progress and resulting insights have been discussed in meetings with industry partners (Syngenta, Rjik Zwaan, East-West Seeds, Mars), at national and international scientific meetings (Plant Genome Stability and Change 2018 (Gatersleben, Germany), in presentations for Seed Central here at UCD (Asta Meets UC Davis and Tomato Processing Conference), and with potential collaborators at other universities. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Several undergraduates and junior researchers have been trained in molecular genetics and plant genetics as part of this project. How have the results been disseminated to communities of interest?Our progress and resulting insights have been discussed in meetings with industry partners (Syngenta, Rjik Zwaan, East-West Seeds, Mars), at national and international scientific meetings (Plant Genome Stability and Change 2018 (Gatersleben, Germany), in presentations for Seed Central here at UCD (Asta Meets UC Davis and Tomato Processing Conference), and with potential collaborators at other universities. What do you plan to do during the next reporting period to accomplish the goals?1) Perform long read sequencing of the desireee CENH3 alleles, followed by characterization of our CRISPR-treated lines. 2) Test tomato in-frame deletions for the ability to induce haploids, further develop transgenic stocks, investigate transmission of KO alleles.
Impacts
What was accomplished under these goals?
To review our goals: Plants carrying mutations in the gene encoding CENH3 can result in haploid induction on outcrossing to plants wild-type for CENH3, resulting in uniparental haploids derived only from the wild-type pollen's genome. We will create lines of tomato and potato that have lost the wild-type CENH3 allele and carry instead a modified CENH3. This requires: A) Elimination of the wild-type allele via CRISPR technology, and simultaneously... B) Complementation of the (essential) CENH3 function by a mutant allele. We will complement the CENH3 knockout allele with two very different classes of CENH3 mutants: --Alleles encoding subtle, single amino acid changes shown to induce haploids at approximately 15% of progeny in Arabidopsis --A more elaborate, chimeric, transkingdom fusion protein that produces haploids in the majority of Arabidopsis progeny. C) These plants will then tested for their frequency of haploid induction. During the last year, our potato work progressed very slowly. Transgenic lines carrying transgenic mutant alleles have been propagated, but we have encountered difficulties in characterizing CRISPR generated mutations at the native locus, as the stock- Desiree- is tetraploid and highly heterozygous. This year we will work to obtain the long sequencing reads required for determination of haplotypes at this locus. We have generated several CRISPR mutant lines of tomato. We have not yet determined whether our chimeric tomato construct (citrine:tailswap) can complement a knockout of the native CENH3. We have developed lines of tomato that carry in-frame small deletions of CENH3 at the native locus. These lines are unexpectedly viable and fertile, and we will test these to see if they can induce haploids, with or without complementing transgenes. We have discovered that similar deletions do induce haploids in Arabidopsis. This type of potentially haploid-inducing CRISPR-induced CENH3 allele is the subject of our recent patent application.
Publications
|
Progress 10/01/16 to 09/30/17
Outputs
Target Audience:Our progress and resulting insights have been discussed in meetings with industry partners (Syngenta, Rjik Zwaan, East-West Seeds, Mars), at national and international scientific meetings (Solgenomics 2016 (Davis), Accelerated Crop Breeding (Cold Spring Harbor, 2016), in a presentation for Seed Central here at UCD, and with potential collaborators at other universities. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?These projects are headed up by two staff scientists; both scientists supervise undergraduate interns. These interns received training in: overall strategies of plant breeding, techniques of molecular biology (PCR, sequencing, in silico analysis of DNA sequences, construction of plasmids, genotyping of plants), concepts of genetic engineering, and plant crossing and propagation. How have the results been disseminated to communities of interest?see "target audiences" above. What do you plan to do during the next reporting period to accomplish the goals?1) T0 (newly transformed) plants will be genotyped for the presence of mutations at CENH3 and for the presence or absence of transgenes. 2) Plants with promising mutations (some already in-hand) will be self-pollinated and outcrossed to produce derivatives carrying the desirable non-null CENH3 mutations in the absence of transgenes and to test for heritability of these CRISPR-generated mutations. 3) Plants homozygous or biallelic for these mutations will be tested for haploid induction. The ability of the chimeric transgene citrine-tailswapCenH3 to complement a cenH3 null will be investigated.
Impacts
What was accomplished under these goals?
To review our goals: Mutations in the gene encoding CENH3 can result in haploid induction on outcrossing to plants wild-type for CENH3, resulting in haploid derived soly from the wild-type pollen's genome. We will create lines of tomato and potato that have lost the wild-type CENH3 allele and carry instead a modified CENH3. This requires: A) Elimination of the wild-type allele via CRIPR technology, and simultaneously... B) Complementation of the (essential) CENH3 function by a mutant allele. We will complement the CENH3 knockout allele with two very different classes of CENH3 mutants: --Alleles encoding subtle, single amino acid changes shown to induce haploids at approximately 15% of progeny in Arabidopsis --A more elaborate, chimeric, transkingdom fusion protein that produces haploids in the majority of Arabidopsis progeny. C) These plants will then tested for their frequency of haploid induction. During the review period, Several tomato lines were generated via CRISPR /CAS mutagenesis that carry potential null or haploid-inducing alleles at CENH3. A similar approach was employed to create potato lines that potentially carry similar important new alleles- these lines have not yet been characterized. Additional tomato transformations applying slightly different approaches to mutagenesis and/or transgenic complementation of CENH3 function were also generated and await characterization.
Publications
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