Source: WASHINGTON STATE UNIVERSITY submitted to NRP
UNDERSTANDING AND MANIPULATING CHROMOSOME-PAIRING CONTROL FOR POLYPLOID WHEAT IMPROVEMENT
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1006750
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Jul 3, 2015
Project End Date
Mar 31, 2019
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
WASHINGTON STATE UNIVERSITY
240 FRENCH ADMINISTRATION BLDG
PULLMAN,WA 99164-0001
Performing Department
Crop & Soil Sciences
Non Technical Summary
Hexaploid wheat is not only the world's most important food crop but is also a model system for the analysis of the basic genome evolution and speciation mechanisms as well as allopolyploid crop improvement approaches. For consistent improvement in wheat yield and quality, a continual search for new genes/gene combinations and their introgression into adapted material is required. Also required is an efficient method of transferring and manipulating genes without undesirable carry-over chromatin.About 50% of all higher eukaryotes including important crop plants are polyploids. Unlike in diploids, more than two identical (homologs) or similar (homoeologs) chromosomes are available for meiotic pairing. Regulation of chromosome pairing is the key step in recovering a sexually propagating, diploidized, hence, fertile polyploid species. In the absence of chromosome pairing control, both homologs as well as homoeologs can potentially pair resulting in an irregular pairing and multivalent formation, giving rise to aneuploidy and thus, sterility. In order to recover reproductively stable polyploid a precise chromosome pairing regulatory mechanism is essential. Chromosome pairing regulator genes have been identified in wheat and many other species. Of these the Ph1 of wheat is the most prominent and perhaps the best characterized. This project is focused on characterizing the Ph1 gene, and manipulating it to develop a targeted method of alien introgression.The project will specifically address the following questions: i) how do the chromosome pairing regulator genes operate to distinguish homologs from homoeologs, ii) during which stage of meiosis are these genes active, iii) how do these genes fit into the current model of chromosome pairing and recombination, iv) are these genes functional only in fertile polyploid species or are they universally present, and v) what is the relationship between the gene with similar genes in other polyploids and with similar function genes such as mismatch repair genes. Currently, we do not have clear answers to any of these questions in any eukaryotic species. In addition to the above-mentioned novel information, characterization of the Ph1 gene will have a direct application in alien introgressions.The wild relatives of crop plants possess a wealth of genes useful for crop improvement. Ideally, only one or a few gene transfers are desirable from otherwise agronomically undesirable wild relatives. At present one/few gene transfers from wild relatives can be most efficiently accomplished by crossing-over between the chromosomes of cultivated and alien species. Because of the presence of Ph1-like genes, such crossing-over is restricted. As a result, many undesirable traits are also transferred along with the desirable genes. In wheat, mutants can be used to activate homoeologous chromosome pairing. Difficulty in the gene scoring has been one of the bottle-neck steps for such manipulations. The project outcome about the mode of action of the Ph1 gene may suggest novel approaches for such alien gene transfers in crop plants in general.
Animal Health Component
40%
Research Effort Categories
Basic
50%
Applied
40%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011549108080%
2062499104020%
Knowledge Area
206 - Basic Plant Biology; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1549 - Wheat, general/other; 2499 - Plant research, general;

Field Of Science
1040 - Molecular biology; 1080 - Genetics;
Goals / Objectives
The long-term goal of the proposed project is to understand role of the Ph1 gene in the process of homology search and chromosome pairing; and to use it for targeted alien introgression. The proposed project will confirm that the C-Ph1 is indeed the Ph1 gene along with studying its mode of action at the molecular level. Additionally, a targeted alien introgression method using the C-Ph1 gene will be optimized which will also confirm the identity of the gene in addition to revolutionizing the alien introgression in wheat and perhaps in other crop plants. Specific objectives of the proposed project are to:1. Perform gene complementation of the Ph1 gene mutants (ph1b and ph1c) with the C-Ph1 by agrobacterium mediated transformation;2. Study evolution of the Ph1 gene and its role in determining the fate of polyploid genomes;3. Study role of the C-Ph1 in homology search and chromosome pairing; and4. Optimize targeted alien introgression by manipulating the C-Ph1.gene.
Project Methods
Objective# 1. Gene complementation of Ph1 gene mutants: To further substantiate our claim that the C-Ph1 is the Ph1 gene, we will perform complementation of the ph1b and ph1c mutants. The 5B copy of the C-Ph1 will be expressed under its resident promoter in wheat cv. Bobwhite. The transgene will then be transferred to the mutants by crossing. Transformation will also be attempted in ph1b and ph1c. We have a reliable and user-friendly method of wheat transformation working in our lab that generates transformants at an efficiency of 1-5% (explant basis). Though other transgenic methods like particle bombardment are also available, but agrobacterium mediated wheat transformation is the method of choice. Transferring DNA via agrobacterium results in a lower transgene copy number and stable integration with fewer rearrangements (Jones and Pašakinskien?, 2005; Dai et al., 2001). Our results have shown that the B-copy of the C-Ph1 gene encodes two transcript variants, each possibly have a different function (Bhullar et al., 2014). Each variant of the B-copy of the gene will be individually expressed and transferred to the mutants. The transgenes will be characterized for the copy number, gene expression, and chromosome pairing analysis using our standard protocols. Normal diploid-like pairing in the mutant plants after transferring the transgenes will confirm that the C-Ph1 is indeed the Ph1 gene. The transgenes will also be evaluated for other phenotypes including early stages of chromosome pairing, centromere clustering, chromosome-microtubule interaction, and interlocking of the bivalents, in order to confirm involvement of the C-Ph1 gene in these processes, as well as to study various functions of the 5B-copy variants.Wheat transgenic lines carrying the 5B-copy variants of the C-Ph1 gene similar to that proposed above but with the GFP tag, will also be made using the 5B-copy promoter. This line will help localize the Ph1 gene protein in the cell and to validate the antibody results. This experiment will also be a backup in case the antibody yields cross-reactivity. These transgenic lines will also make 'live' tracking of the C-Ph1 gene protein(s) in the cell possible, and will show its possible interactions with telomeres, centromeres, pairing centers, or microtubules.Objective# 2. Evolution of the C-Ph1 gene: For understanding the evolution of the Ph1 gene, structure and expression of the C-Ph1 gene will be studied in the wheat tetraploid and diploid ancestors viz. Triticum urartu, Aegilops speltoides, Aegilops tauschii, Triticum teemopheevi, Triticum araraticum, Triticum turgidum, and Triticum dicoccum. These species have been extensively studied for their chromosome pairing behavior. The C-Ph1 gene orthologs identified from the genomic and/or EST sequences from Triticum monococcum, A. tauschii, and T. turgidum showed a very high sequence similarity with the corresponding wheat copies, indicating the presence of the gene in the ancestral species as well. To understand how the gene evolved during the course of evolution especially around the polyploidization steps, gene structure and expression pattern of the C-Ph1 gene will be studied in these species. The full-length genomic and cDNA copies of the gene will be cloned from two or more accessions of each of these species by PCR amplifying the corresponding sequences using the C-Ph1 gene-specific primers from wheat (Bhullar et al., 2014). The 5B copy of the gene seems to have evolved novel function of differentiating homologs from homoeologs by an insertion of 60 bp, alternate splicing, and/or specific expression of the gene copy during metaphase I. Structure and sequence of the C-Ph1 gene orthologs from each of the ancestral species will be compared to study the evolutionary timeline and role of each of these changes in the function of the 5B copy. Additional structural and expression differences including deletions, insertions, and intron-exon splice junctions will also be evaluated for their possible role in the evolution of the Ph1 gene function and chromosome pairing in general.Detailed bioinformatic analysis of the C-Ph1 gene copies will be carried out to identify domains, motifs, and regulatory elements present in the gene and its promoter. The gene sequences both at the DNA as well as protein level will be scrutinized for the presence of known or novel motifs with possible role in centromere, telomere, or microtubule binding. The three copies of the gene will be compared in detail to identify subtle differences possibly involved in the novel function of the 5B copy. Since the expression of the 5B copy of the gene is very specific to the metaphase I stage of meiosis, effort will be made to identify the regulatory element(s) providing this specificity. This will be performed by comparing the expression pattern of the gene among various species, with the presence of various regulatory elements. The regulatory elements identified from this analysis will be the target for the CRISPR analysis if that works in wheat. Dr. von Wettstein is currently optimizing this approach in wheat (see later section).Objective# 3. Study role of the C-Ph1 gene in homology search and chromosome pairing: To study if the silencing of the C-Ph1 gene results in homoeologous pairing and/or non-homologous pairing, wheat-rye disomic substitution lines for chromosome 1B(1R) and monosomic substitution line for chromosome 5B(5R) will be crossed. The F1 plants carrying the 5R chromosome will be selected by using 5R specific markers or by chromosome root-tip analysis. VIGS will be performed using our lab protocol. Meiotic chromosome pairing analysis will be performed on the F1 plants carrying the C-Ph1 gene and will be compared to the plants lacking the gene. Chromosomes 1B and 5B are easily distinguishable based on NOR (nucleolus organizer region) and arm ratio, thus can be easily monitored. The C-banding analysis will also be performed to differentiate if the chromosome pairing is happening only among homoeologous in the absence of the C-Ph1 gene or the pairing also occurs among non-homologs. Expression of the different copies of the C-Ph1 gene will be studied in the cells at various meiotic stages in order to determine expression pattern differences. Poly-clonal antibodies will be developed both from synthetic peptides of the selected candidate gene as well as using the protein expressed in E. coli. The antibodies from the peptides have already been ordered (commercially prepared), and the gene protein purification from E. coli is underway in the lab. The antibodies will be used for western blots to study proteins and their modifications. The antibodies will also be used to localize the C-Ph1 gene protein during various stages of mitotic and meiotic cell division to determine its specific role in each of these two processes. Special attention will be placed on studying possible interaction of the gene with centromeres, telomeres, microtubules, and other cell components. Immuno-localization will be performed during various stages of meiosis and mitosis to determine mode of action of the gene.Objective# 4. Optimize targeted alien introgression by manipulating C-Ph1: With the objective to test if the C-Ph1 gene can be used to induce recombination between wheat and its wild relatives, the C-Ph1 gene will be silenced by VIGS in the plants heterozygous for 1B/1R translocation in the wheat cultivar Pavon background. Pollen from these plants will be used to pollinate wheat cultivar Louise to generate a population of about 500 plants. The population will be screened for the plants carrying the stripe rust resistant gene (present on chromosome 1R of rye) but otherwise lacking the rye chromosome. Progeny of the plants after VIGS will be evaluated for recombination as well as for chromosome pairing. Effect of the C-Ph1 gene silencing on homoeologous as well as homologous recombination will be studied.

Progress 07/03/15 to 03/31/19

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The project has been terminated early due to the consolidation of research projects into collaborative groups. This goal and the objectives have been incorporated into Hatch umbrella project WNP00008 (Accession# 1015621) and the accomplishments are being reported under that project.

Publications


    Progress 10/01/17 to 09/30/18

    Outputs
    Target Audience:Scientific community, farmer organizations, students, public at large Changes/Problems:Part of the project activiteis have been now added to the new umbrella hatch project: WNP00008, Accession# 1006750 What opportunities for training and professional development has the project provided?a. Two PhD students were able to go to two scientific meetings for professional development and to present their results. b. Three post docs were trained along with three graduate students and three undergraduate students.? How have the results been disseminated to communities of interest?Publications in peer reviewed journals Invited and volenteered seminars at the scientific meetings, universities, research institutes. Grower group interactions Workshops and training sessions. What do you plan to do during the next reporting period to accomplish the goals?1. Study evolution of the Ph1 gene and its role in determining the fate of polyploid genomes; 2. Study role of the C-Ph1 in homology search and chromosome pairing; and 3. Optimize targeted alien introgression by manipulating the C-Ph1.gene.

    Impacts
    What was accomplished under these goals? a. To establish in which species the Ph1 gene evolved. b. Specifically studying the structural changes differentiating the 5B copy of the gene from the 5A and 5D copies, we studied which of the structural and expression differences are responsible for the evolution of the precise chromosome pairing control. c. To explore if the C-Ph1 gene can be used to induce chromosome pairing between wheat and its wild relative chromosomes to result in alien gene transfer. d. The complementation experiment was conducted to establish if the C-Ph1 gene can completely restore the gene function in the Ph1 gene mutations. e. Since the C-Ph1 gene has a novel expression pattern with dramatic changes in transcript accumulation, the promotor study was conducted to identify DNA motifs and/or elements responsible for the novel expression pattern. 3) significant results achieved, including major findings, developments, or conclusions (both positive and negative): a. We have shown that the Ph1 gene evolved in the emmer wheat after the polyploidization event. b. The structural features of the 5B copy are not sufficient for the chromosome pairing control but is required. c. The three splice variants of the C-Ph1 gene show differential accumulation during various meiotic stages. d. Dramatic increase in the accumulation of one of the three transcripts is responsible for the chromosome pairing control. e. Since the structural changes in the 5B of the gene results in the three splice variants, both the structural changes and the meiosis specific differential accumulation of one of the 5B transcripts is responsible for the chromosome pairing control. 4) key outcomes or other accomplishments realized. a. Based on the preliminary data, we were able to induce recombination between wheat and rye chromosomes by transiently silencing the C-Ph1 gene via virus induced gene silencing. b. We have identified promotor elements that maybe responsible for the novel expression of the 5B copy of the gene.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2018 Citation: Kanwardeep Singh, Johar Singh, Suruchi Jindal, Gaganjot Sidhu, Amandeep Dhaliwal and Kulvinder Gill 2018. PIN1 structural and functional evolution across monocots and dicots. Functional and Integrative Genomics https://doi.org/10.1007/s10142-018-0625-9
    • Type: Journal Articles Status: Published Year Published: 2018 Citation: Nathan P. Grant. Amita Mohan, Devinder Sandhu, and Kulvinder S. Gill 2018. Inheritance and genetic mapping of the reduced height (Rht18) gene in wheat. Molecular Plant Breeding 7, 58; doi:10.3390/plants7030058
    • Type: Journal Articles Status: Published Year Published: 2017 Citation: Ragupati Nagarajan and Kulvinder S. Gill 2017. Evolution of Rubisco activase gene in plants. Plant Molecular Biology https://doi.org/10.1007/s11103-017-0680-y
    • Type: Journal Articles Status: Published Year Published: 2017 Citation: Bhavya P. Mishra, Rajeev Kumar, Amita Mohan, and Kulvinder S. Gill 2017. Conservation and divergence of Starch Synthase III genes of monocots and dicots. PLOS ONE https://doi.org/10.1371/journal.pone.0189303


    Progress 10/01/16 to 09/30/17

    Outputs
    Target Audience:Scientists, stakeholders, administrators and public at large. Changes/Problems:We continue to seek additional funding for this project but so far no success. We just submitted a project to NSF to generate additional support for the project. What opportunities for training and professional development has the project provided?One PhD student working on this project (Ahsan Khan) recently completed his preliminary examination. Other PhD students, Kanwardeep Singh and Karansher Singh Sadhu are also working on the project along with contributions from a Post Doctorate fellow (Ramanjot K. Bhullar), who initially completed her PhD working on the project.Few other visiting scientists have also assisted in completing few of the objectives of the project. Because of the popularity of the gene and the story, I have been invited to deliver numerous talk on the story and has resulted in atleast two new collaborations. How have the results been disseminated to communities of interest?Original story was published in a very high impact factor journal (PNAS) and a followup paper is under review in another very high impact factor journal (The Plant Cell). Other manuscripts are in the submission stage including comparative analysis of Ph1 gene across other species and an opinion paper on homology search in polyploids. The story about the gene was covered by various major news outlets including Yahoo news. A patent has been issued on the use of the gene to induce recombination between wheat and wild relatives. What do you plan to do during the next reporting period to accomplish the goals?We will continue to understand mechanism of the gene especially during the homology search process. We will further work to understand the evolution of Ph1 gene and role it played in wheat evolution. We will also work on testing the utilization of this highly specific promoter for hybrid seed development. Finally, we will publish the work on the evolution of Ph1 gene and its applicability for alien gene transfers.

    Impacts
    What was accomplished under these goals? Previously we reported C-Ph1 as a candidate for the wheat chromosome pairing regulator gene, Ph1, that controls diploid-like pairing behavior of wheat by differentiating homologous from homoeologous and non-homologous chromosomes . Here, we report that the ph1b mutant lines, when complemented with the C-Ph1 gene exhibited very low levels of homoeologous pairing when crossed with rye thus confirming the identity of the gene. Additionally, we report that the 5B copy of the gene evolved its novel function of differentiating homologs from homoeologs via unique changes in promoter leading to 457-fold higher expression during prophase I, acquisition of novel motifs, specific insertions/deletions, and tissue-specific retention of introns. The three gene copies that putatively make at-least seven protein variants also show dramatically different expression patterns indicating possibility of many different functions associated with the Ph1 gene. One of these putative proteins (5B80) showed 3D structure similarity with the RuvA-RuvB motor protein complex required for the Holliday junction branch migration and other similar processes, and the transcript coding of this protein showed the highest expression during the stages where such motor protein complexes are required. We have also also investigated the structural and putative functional organization of the gene across other species. Identified from seven monocots and three dicots using specific and stringent criteria, the C-Ph1 gene's 'true' orthologs showed a typical pattern of three exons and two introns similar to that of the 5B copy. However, these showed a higher sequence identity with the 5D copy at the predicted amino acid level suggesting it to be the conserved and ancestral version of the gene. With unprecedented higher expression at meiosis, the gene orthologs showed a similar expression pattern as that of the C-Ph1 gene thus confirming the functional conservation of the gene across different plant genera to regulate the basic process of chromosome pairing. Further, Ph1 gene of wheat has been used to induce recombination between cultivated wheat and its wild relatives. Presence of Ph1 gene restricts the crossing over between wheat and alien chromosomes, thus limiting the possibility of gene transfers from wild relatives for biotic and abiotic stress tolerance. In wheat, Ph1 mutants such as ph1b and ph1c can be used to activate/allow homoeologous chromosome pairing but the problem with this approach is the difficulty in gene scoring in segregating generations. We developed a transient approach of silencing Ph1 gene using Virus induced gene silencing to induce recombination in wheat. cv Pavon 1BL.1RS translocation was crossed with Zak 1BL.1BS and gene silencing was performed on F1's. Recombination was studied in segregating backcross population and ~20% recombinants were found in VIGS population compared to control population where only 1% recombinants were scored. Using a simple approach we have delimited the core promoter region of the gene and have identified promoter elements possibly responsible for the expression specificity. Unique expression of Ph1 gene is key to its function as seen during functional characterization using RNAi, where extent of gene silencing correlated with the severity of phenotype. Thus, to delineate the region of Ph1-5B controlling the specific expression during meiosis. We developed a similarity plot matrix to delimit the core region required for gene expression. For validation and confirmation of the core promoter, We made four deletion series (DS) constructs corresponding to DS1 of 900bp, DS2 of 625bp, DS3 of 500bp and DS4 of 250bp promoter sequence upstream of first start codon. These constructs were cloned in pCAMBIA 1305.1 plasmid vector containing a GUSplus gene for this experiment and the 35S promoter was replaced with the 5B promoter deletion series. The constructs were sequence confirmed, and transferred into wheat cv. bobwhite using Agrobacterium mediated genetic transformation.T0 transgenics were confirmed using qPCR and atleast five independent transgenics were selected for each deletion series. GUS activity assay is underway.

    Publications

    • Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Nagarajan, Ragupati and Kulvinder S. Gill 2017. Evolution of Rubisco activase gene in plants. Plant Molecular Biology (In press). DOI: 10.1007/s11103-017-0680-y
    • Type: Journal Articles Status: Published Year Published: 2017 Citation: Jasdeep S. Mutti, Ramanjot Bhullar and Kulvinder S. Gill 2017. Evolution of Gene Expres- sion Balance among Homoeologues of Natural Polyploids. G3: Genes Genomes Genetics 7: 1225-1237. doi.org/10.1534/g3.116.038711
    • Type: Journal Articles Status: Published Year Published: 2017 Citation: Simerjeet Kaur, Xu Zhang, Amita Mohan, Haixiao Dong, Prashant Vikram, Sukhwinder Singh, Kanwarpal Singh Dhugga, Zhiwu Zhang, Kulvinder S Gill, Jaswinder Singh 2017 Genome-wide association study reveals novel genes linked to natural variation of culm cellulose content in bread wheat (Triticum aestivum, L.). Frontier Plant Sci., 06 November 2017 https://doi.org/10.3389/fpls.2017.01913
    • Type: Journal Articles Status: Published Year Published: 2017 Citation: N. Kumar, Harpinder S. Randhawa, Ryan W. Higginbotham, Xianming M. Chen, Timothy D. Murray, and Kulvinder S. Gill. 2017. Targeted and efficient transfer of multiple value-added traits into wheat varieties. Molecular Breeding 37:68. DOI: 10.1007/s11032-017-0649-1
    • Type: Journal Articles Status: Published Year Published: 2016 Citation: Marwa N. M. E. Sanad, Kimberley Garland Campbell and Kulvinder S. Gill 2016. Develop- mental program impacts phenological plasticity of spring wheat under drought. Botanical Studies 57: 35. DOI 10.1186/s40529-016-0149-3.
    • Type: Journal Articles Status: Under Review Year Published: 2017 Citation: Aiqing SunImpa, M Somayanda, John Sunoj, Kanwardeep Singh, P.V. Vara Prasad, Kulvinder Gill, S.V. Krishna Jagadish 2017 Heat stress during flowering affects time of day of flowering, seed-set and grain quality in spring wheat (Triticum aestivum L.). Crop Science (under review)
    • Type: Journal Articles Status: Under Review Year Published: 2017 Citation: Raju Bheemanahalli, V.S. John Sunoj, Gautam Saripalli, P.V. Vara Prasad, H.S. Balyan, P.K. Gupta, Kulvinder S. Gill, and S.V. Krishna Jagadish 2017 Quantifying the impact of heat stress on pollen germination, seed-set and grain filling in spring wheat. Crop Science (under review).
    • Type: Journal Articles Status: Submitted Year Published: 2017 Citation: Nathan Grant, Amita Mohan, Devinder Sandhu and Kulvinder S. Gill 2017. Inheritance and genetic mapping of the reduced height (Rht18) gene in wheat. PLoS One (submitted).


    Progress 10/01/15 to 09/30/16

    Outputs
    Target Audience:Scientists, Peers, Stakeholders Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One PhD student (Ramanjot Bhullar) completed her PhD working on this project and is now working as a post doc. Now another student Ahsan Khan is working on the project along with some contribution from Kanwardeep Singh and few visiting scientists. Because of the popularity of the gene and the story, I have been invited to deliver numerous talk on the story and has resulted in at least two new collaborations. How have the results been disseminated to communities of interest? Original story was published in a very high impact factor journal (PNAS) and a followup paper is under review in another very high impact factor journal (The plant Cell). The story about the gene was covered by various major news outlets including Yahoo news. A patent is pending on the use of the gene into induce recombination between wheat and wild relatives. What do you plan to do during the next reporting period to accomplish the goals?We will continue to understand mechanism of the gene and will work on testing if we can use the gene to transfer value-added genes from wild relatives into cultivated wheat. Evolution of the Ph1 gene will also be studied.

    Impacts
    What was accomplished under these goals? Previously we reported C-Ph1 as a candidate for the wheat chromosome pairing regulator gene, Ph1. Here we report that the 5B copy of the gene evolved the novel function of differentiating homologs from homoeologs via unique changes in promoter leading to 457-fold higher expression during prophase I, acquisition of novel motifs, specific insertions/deletions, and tissue-specific retention of introns. The three gene copies putatively make at-least seven protein variants with different expression patterns possibly evolving many different functions associated with the Ph1 gene. One of these putative proteins (5B80) structurally resembles the RuvA-RuvB motor protein complex required for the Holliday junction branch migration and other similar processes, and showed the highest expression during the stages where such motor protein complexes are required. Using a simple approach we have delimited the core promoter region of the gene and have identified promoter elements possibly responsible for the expression specificity. Hexaploid wheat is a relatively 'young' polyploid with intact homologous (similar) and homoeologous (related, from different genomes) chromosomes that are highly similar in gene content and synteny. Despite pairing potential of the homoeologs, wheat behaves like a diploid mainly due to the presence of the chromosome pairing control exerted by the Ph1 (Pairing homoeologous 1). Originally discovered in 1958 in a wheat line lacking chromosome 5B, the Ph1 locus was localized to a ~2.5Mb region on the long arm of the chromosome 5B ('Ph1 gene region') bracketed by deletion breakpoints. No point mutation is available for the gene although a deletion mutant in hexaploid (ph1b) and tetraploid (ph1c) wheat is available, each involving an interstitial deletion of ~1µm. The 5B copy of the Ph1 gene is the predominant regulator of homoeologous chromosome pairing (HECP) as its absence results in higher order pairing or multivalent formation during meiotic metaphase I (MI). While, the absence of chromosome 5A had essentially no effect on HECP whereas the lack of 5D chromosome exhibited asynapsis, frequent bivalent interlocking and rare multivalents. The lack of 5D effect on HECP was not as robust as was observed in the absence of chromosome 5B. Asynapsis was not observed in the absence of either 5A or 5B chromosomes. Double dose of neither 5A nor 5D was able to restore normal HECP, disrupted in the absence of chromosome 5B. Similarly, extra doses of 5B was unable to compensate for the loss of 5D copy, evoking the divarication of the two homoeologs in addition to some overlapping functions. Minimum four doses of the 5A copy were needed to compensate for the loss of the 5D copy, suggesting a common function of the two with the former having a weaker effect. Six copies of the 5BL chromosome arm resulted in bivalent interlocking along with rare multivalents and reduced chiasmata frequency even among homologs while normal chromosome pairing was observed in the presence of extra doses of 5A or 5D chromosomes. These along with some other observations suggest that the Ph1 gene copies on the three homoeologs have evolved significantly different with slightly overlapping functions during the course of evolution. In addition to HECP, the 5BL copy of the Ph1 locus has been implicated in several other meiotic functions. Measured as a sensitivity of meiotic spindle to anti-microtubule drugs, the loss of chromosome 5B was shown to affect dynamics of spindle assembly, affecting microtubule-centromere interaction via altered phosphorylation of tubulin proteins. Similar results on the centromere microtubule-interactions were reported when analogous experiments using the ph1b mutation were performed, localizing the observed affect to a ~1µm region of chromosome 5B that is deleted in ph1b. Involvement of the 5B copy of the Ph1 gene in centromere function was further supported by the observation of significantly altered transverse division of univalents in the ph1b mutant. Furthermore, chromosomal arrangements during the pre-meiotic mitosis and chromosomal movement towards the poles was significantly different in the ph1b mutant. Loss of chromosome 5B also resulted in reduction of both speed and degree of chromosome condensation during cell division. Additionally, it resulted in aberrant morphology of the rye telosomes present in the wheat background, disturbed pre-meiotic associations, and reduced and irregular pairing during zygotene and pachytene. Similarly, premature and asynchronous remodeling of chromatin was shown in wheat-rye hybrids in the presence of Ph1 gene. Taken together these reports suggest that either the Ph1 locus carry a single gene with homoeologs having different and some overlapping functions, or the locus has a cluster of genes involved in meiosis related functions. Recently we identified C-Ph1, silencing of which resulted in HECP similar to that observed in the absence of the Ph1 locus. Here we show that the three structural C-Ph1 gene copies make at least five transcripts via alternate splicing, resulting in seven putative proteins. The 5B copy of the gene contains a novel 60bp insertion and a 29bp deletion resulting in three splice variant mRNAs coding for five putative proteins. One of these putative proteins (5B80) showed strong structural resemblance to the specialized motor proteins involved in Holliday junction branch migration and other similar processes to regulate HECP. Unlike other variants, this novel putative protein specifically expresses during prophase I due to tissue-specific intron retention and unique changes in the 5B copy promoter.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2019 Citation: Marwa N. M. E. Sanad, Kimberley Garland Campbell and Kulvinder S. Gill 2016. Developmental program impacts phenological plasticity of spring wheat under drought. Botanical Studies 57: 35. DOI 10.1186/s40529-016-0149-3.
    • Type: Journal Articles Status: Published Year Published: 2016 Citation: Ritu Batra, Gautam Saripalli, Amita Mohan, Kulvinder Singh Gill, Harindra Singh Balyan, and Pushpendra Kumar Gupta 2016. Comparative structural and functional analysis of AGPase in monocots and dicots. Frontiers in Plant Science 8:19. DOI:10.3389/fpls.2017.00019
    • Type: Journal Articles Status: Published Year Published: 2016 Citation: Sethi Sorabh, Johar S. Saini, Amita Mohan, Navreet K Brar, Shabda Verma, Navraj K Sarao, and Kulvinder S Gill 2016. Comparative and evolutionary analysis of alpha-amylase gene across monocots and dicots. Functional and Integrative Genomics 16 (5): 545-555.
    • Type: Journal Articles Status: Published Year Published: 2016 Citation: Reynolds MP, Quilligan E1, Aggarwal PK, Cavalieri A, Chapman SC, Chapotin SM, Datta SK, Gill KS, Jagadish KSV, Koehler AK, Kosina P1, Lafitte R, Mahala RS, Paterson AH, Prasanna BM, Rosegrant MW, Sharma I, Sivasankar S, Vadez V, Valluru RI, Vara Prasad PV, Yadav OP 2016. An integrated approach to maintaining cereal productivity under climate change. Global Food Security 8: 9-18.
    • Type: Journal Articles Status: Published Year Published: 2016 Citation: Simerjeet Kaur, Kanwarpal S Dhugga, Kulvinder Gill, Jaswinder Singh 2016. Comparative analysis of cellulose synthase (CESA) genes reveals novel structural and functional conservation in hexaploid wheat. PLOS ONE 11 (1), e0147046
    • Type: Journal Articles Status: Accepted Year Published: 2016 Citation: N. Kumar, Harpinder S. Randhawa, Ryan W. Higginbotham, Xianming M. Chen, Timothy D. Murray, and Kulvinder S. Gill. 2017. Targeted and efficient transfer of multiple value-added traits into wheat varieties. Molecular Breeding (accepted).
    • Type: Journal Articles Status: Submitted Year Published: 2016 Citation: D. Dewan, N. Kaur, K. S. Gill, M Sahni, A. P. Chopra, A. B. Pant, and G. S. Randhawa 2017. Identification and functional characterization of rootless concerning crown and seminal roots (RTCS) gene of wheat. Journal of Experimental Botany (Submitted).
    • Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Nilesh Shirish Wagh, Navin Chander Gahtyari, Amita Mohan, Kulvinder Singh Gill, Jai Prakash Jaiswal and Gajanan Balasaheb Zore 2017. Structural and Functional Evolution of Sucrose Synthase among Monocots and Dicots. PloS Computational Biology (Submitted).
    • Type: Journal Articles Status: Other Year Published: 2016 Citation: Jasdeep S. Mutti, Ramanjot Bhullar and Kulvinder S. Gill 2017. Evolution of Gene Expression Balance among Homoeologues of Natural Polyploids. G3: Genes Genomes Genetics (Submitted after revision).
    • Type: Journal Articles Status: Under Review Year Published: 2016 Citation: Ramanjot Bhullar, Kanwardeep Singh, Muhammad Khan, Amita Mohan, and Kulvinder Gill 2017. Unique expression pattern, insertions/deletions and alternate splicing evolved the novel function of the Ph1 gene of wheat. The Plant Cell (Under review).
    • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Kulvinder Gill 2016. Climate resilient wheat varieties for the changing climate. ICSAP, Faisalabad Pakistan, Nov 17 to 19 (Invited plenary talk).
    • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: ulvinder Gill, Amita Mohan, Neeraj Kumar, Kanwardeep Singh, Nathan Grant, Ragupathi Nagarajan, Suman Bakshi, Johar Singh, P.V. Vara Prasad, Jai Jaiswal, Pushpendra K. Gupta, Rajeev Kumar, Sindhu Sareen, Hoshiyar Singh, Harindra S. Balyan, K K. Narayanan, Viswanathan Chinnusamy and Rakesh Singh 2016. Developing Heat Tolerant and Climate Resilient Wheat. New approaches in biotechnology and biosciences, Feb 18-20, 2016, Agra, India.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Jake Lazar, Sheila Fitzgerald, Ragupathi Nagarajan and Kulvinder Gill 2016. Identification of mechanisms controlling heat tolerance in wheat. SURCA, WSU.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Alexander Blackburn, Gaganjot Sindhu, Amita Mohan, Kulvinder S Gill 2016. Molecular mapping of genes controlling seedling emergence trait in bread wheat. SURCA, WSU.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Leonardo Hinojosa, Neeraj Kumar, Kulvinder Gill and Kevin Murphy 2016. High-throughput phenotyping to evaluate heat stress response in quinoa. Annual Meeting: American Society of Agronomy, Crop Science Society of America, and Soil Science (poster presentation).


    Progress 07/03/15 to 09/30/15

    Outputs
    Target Audience: Scientists, peers, stakeholders. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Training and professional development is an important aspect of the project for which the opportunities were created at all levels. Last year, one graduate student (Ramanjot Kaur) under this project successfully completed her PhD and is now continuing the work and carrying it to the next level by working as a research associate. Currently, there are two PhD students (Kanwardeep Singh and Muhammad Ahsan Khan) who are working on the project and getting training. Last year, one visiting scientist,Dr. Gajanan Zore funded by SRTM university, Nanded, visited WSU for a year. He worked on RNAi transformations and created the transgenics for copy-specific silencing of C-Ph1 gene homoeologs. For undergraduate research, two undergraduates, Trinidad De Jesus and Graham Quinn worked with the RNA and DNA extractions for the project. They also assisted with several other experiments related to the project. They also participated in writing reports and papers and received feedback to improve their scientific writing skills. They also had the opportunity to make presentations at poster sessions at the Western ASPB (American society of plant biologists) held last year in Pullman. Trinidad De Jesus offered oral presentations, with significant practice and coaching beforehand. All the graduate, undergraduate and visiting scientists received training in ethics and responsible conduct of research, which includes topics such as publications and authorship, data acquisition and management, conflicts-of-interest, mentoring, and research misconduct. Three graduate students and a visiting scientist also got the opportunity to present their posters at the western ASPB 2015. One graduate student attended two major scientific meetings, ASPB meeting in Minneapolis and PAG (Plant and animal genome meeting) in SanDiego, thus getting an opportunity for professional development and to present her work at the international level. How have the results been disseminated to communities of interest? The results have been presented at various scientific meetings, field days and other grower meetings. • The results were published in the form of a manuscript in PNAS (PNAS 111(39): 14187-14192). • The press release following the publication got wider publicity including making it to the Yahoo News (http://news.yahoo.com/scientists-discover-breed-non-gmo-super-wheat-genetic-215941041.html). Some of the other news links about the study are given below: Researchers discover new wheat gene http://www.worldgrain.com/articles/news_home/World_Grain_News/2014/09/Researchers_discover_new_wheat.aspx?ID={55EFDA12-4B77470998E1B3325BF7A6D3}&cck=1 Researcher finds gatekeeper wheat gene http://www.capitalpress.com/Research/20140919/wheat-researcher-finds-gatekeeper-wheat-gene Wheat gene discovery clears way for non-GMO breeding https://news.wsu.edu/2014/09/15/wheatgenediscoveryclearswayfornongmobreeding/#.VCjQrktrxLH Discovery allows robust wheat strains without GMO tinkering http://foodenvironmentreportingnetwork.cmail2.com/t/ViewEmail/r/E254A9F1DF8D18C52540EF23F30FEDED/9173ACB2318924E54D402EFBD42943A3 A new process could allow for highly targeted crosses between the domesticated crop and its wild relatives. http://www.takepart.com/feature/2014/09/22/non-gmo-gmo-super-wheat New non-GMO 'super wheat' http://www.digitaljournal.com/news/environment/new-non-gmo-super-wheat/article/404297 Most famous wheat gene' discovered, clears way for non-GMO breeding http://www.sciencedaily.com/releases/2014/09/140915153836.htm Discovery Of The 'Most Famous Wheat Gene' Clears Way To Breed Better, Non-GMO Wheat http://beforeitsnews.com/science-and-technology/2014/09/discovery-of-the-most-famous-wheat-gene-clears-way-to-breed-better-non-gmo-wheat-2720968.html Wheat gene discovery clears way for non-GMO breeding http://www.agprofessional.com/news/Wheat-gene-discovery-clears-way-for-non-GMO-breeding-275169861.html?llsms=978651&c=y 'Most famous wheat gene' found http://phys.org/news/2014-09-famous-wheat-gene.html Companies Pursuing Non-GMO Products For Competitive Edge, Experts Say http://www.ibtimes.com/companies-pursuing-non-gmo-products-competitive-edge-experts-say-1695798 WSU researchers find 'most famous wheat gene' http://www.sciencecodex.com/wsu_researchers_find_most_famous_wheat_gene-141664 Genetically Modified Wheat, Without The GMO Drama http://www.science20.com/news_articles/genetically_modified_wheat_without_the_gmo_drama-144896 • The project was presented in two WSU organized field days attended by 50-200 farmers, politicians, administrators and public at large. • The project was presented twice in the Department of Crop and Soil Science Spring and fall seminar series attended by faculty from various departments, graduates and post-docs. • The project was also presented in the WSU plant science retreat organized every year by the MPS program with support from the Graduate School, the Agricultural Research Center, CAHNRS and the College of Arts and Sciences. In addition to the faculty from various departments, graduates and post-docs, the prospective students of the MPS program are also invited to attend the seminars and poster sessions. • The students and post-docs associated with the project made posters at annual meetings of Western ASPB, ASPB and PAG. • The results from the study were also presented as an invited talk at the annual meeting of the PAG, San Deigo; ASPB, Minneapolis; Western-ASPB, Pullman; Agronomy Society of America, Minneapolis; as an invited seminar at the international durum conference (From seed to pasta and beyond), Milan, Italy; Faisalabad Agriculture University, Pakistan; and at Punjab Agricultural University, India. What do you plan to do during the next reporting period to accomplish the goals? During the next year, a major focus will be to understand the neo-functionalization of the C-Ph1: how did the novel function of differentiating homoeologs from homologs and non-homologs evolve from a gene involved in the basic process of meiosis; detailed molecular characterization of the C-Ph1 gene and its proteins to understand its mode of action; to understand if the 5B80 protein is a specialized-motor protein involved in chromosome pairing and homology search, detailed promoter analysis to identify elements regulating prophase-I/metaphase-I specific expression; and check if C-Ph1 gene can be used to induce recombination. For the last experiment a VIGS based protocol will be optimized. Our current data suggests that one or a combination of the four changes differentiating 5B from other two copies is responsible for evolving the novel function of differentiating homologs from homoeologs during chromosome pairing: i) 29bp deletion; ii) 60bp insertion; iii) Alternate splicing to result in three novel proteins; and iv) Late prophase-I/MI specific expression. We will focus to identify which of these changes are required for this function. For this detailed structural and functional characterization of the C-Ph1 gene in the progenitors of wheat will be done. Our structural and expression analyses suggested that the three copies of the genes and five putative proteins may have many diverse functions during meiosis and perhaps other stages including mitosis. Furthermore, silencing of all three copies of the gene showed other chromosome pairing phenotype in addition to that seen in the Ph1 gene mutations. Taken together the putative proteins of the gene may have multiple different functions thus each copy of the gene needs to be individually characterized. Therefore, in order to study the function of each copy, copy-specific silencing via stable RNAi based transformations will be done. Also, to determine the number of proteins made from the four transcript/variants of the C-Ph1 and for the immunolocalization of the proteins in the cell, western blotting and immunolocalization studies will be done. Our current results suggest that the gene maybe involved in regulating chromosome pairing by modulating centromere function. We will follow that lead to test that hypothesis. The immunolocalization of a-tubulin (antibody for microtubules) and CENH3 (Centromere Histone 3) in the C-Ph1 RNAi silenced plants will be done to understand the role of the gene in regulating centromere-microtubule interactions. Additionally, colocalization of the C-Ph1 protein and CENH3 or a-tubulin proteins at specific subcellular structures will further elucidate the interaction of the C-Ph1 with centromere and microtubule-related proteins. A grant proposal will be submitted to NIH and/or NIFA to obtain funding for this work.

    Impacts
    What was accomplished under these goals? A gene (C-Ph1, candidate for Ph1) was identified, silencing of which resulted in chromosome pairing behavior similar to that of the Ph1 gene mutations including homoeologous chromosome pairing, multivalent formation, and disrupted chromosome alignment on the MI plate. Additional RNAi experiments established that the severity of the chromosome pairing phenotype observed in response to the silencing of the C-Ph1 matched well with the extent of gene silencing thus further strengthening our claim on the Ph1 gene. Chromosome pairing comparison between the VIGS and RNAi lines matched with that of the gene mutant ph1b except that the RNAi and VIGS phenotypes were stronger, probably because the RNAi line has all three copies of the gene silenced compared to only one being deleted in the mutant. As a result of additional RNAi experiments, we managed to identify an RNAi plant, which showed only 44% silencing of the gene and showed a chromosome pairing phenotype similar to that of the Ph1 gene mutations. Full-length gene cloning revealed three copies of the C-Ph1, one each on the three wheat homoeologs that were 90% similar at DNA level. The genomic copy of 5B was the largest (954bp) followed by 5D (883bp) and 5A (539bp) copy. Present 5bp apart, the 5B copy showed an insertion of 46bp and 14bp (referred to as 60bp insertion) that contained an in-frame stop codon. Additionally, it contained a deletion of 29bp. There were two 5D-specific deletions of 12bp and 15bp present in the exon II. The UTRs were 92-97% similar among the gene copies except for the 3' UTR of the 5A copy that did not match with either of the two copies. Excluding insertions and deletions, the 5B copy was 95% similar to the other two copies. Among the three gene copies, 5A produced the smallest transcript. The 5B copy of the gene showed alternate splicing to produce two transcripts of 954bp and 763bp. In addition to the major insertions and deletions, the homoeologs also showed dramatic differences in the form of several single base changes that led to the identification of copy-specific motifs. Three proteins could be predicted from the two 5B transcripts: 80, 204, and 221aa. Protein structural analysis via 3-D modeling also suggested that the gene copies varied significantly. These sequence and structural variations amongst the C-Ph1 gene homoeologs suggested neofunctionalization of the gene. Cumulative expression analysis of all the homoeologs by quantitative real-time expression analysis showed that the gene primarily expresses during the post-flower initiation stages although significant expression was observed in the roots as well. Expression of the gene increased 39-fold in transition from prophase-I to MI. Relative to MI, the expression dropped by 34-fold during anaphase I followed by further drop of 6.4-fold during the dyad stage. Surprisingly, there was 16.5-fold increase in gene expression during the tetrad stage suggesting additional functions of the gene. Copy specific expression analysis revealed that homoeologs have dramatically different expression patterns. With the exception of roots where almost all copies showed expression, the 5B copy specifically expressed during meiosis-I with 23-fold increase in expression from prophase-I to MI in 3-5cm long spike, which in CS contains meiotically dividing cells. Essentially no expression was observed for the copy during meiosis-II and mature anther stages. Expression pattern of the 5D copy was very different from that of the 5B copy. Unlike 5B, the 5D copy showed low-level of expression in the leaves and meiosis-I, but none during anthesis or five days post-anthesis (5DPA). The 5A copy expressed predominantly during meiosis II suggesting its role in cytokinesis and/or gametophyte development The higher level of expression of the 5B copy during late prophase I to MI coincides with the stage when the homologous pairing is suggested to be differentiated from homoeologous pairing. The expression differences among the C-Ph1 gene homoeologs strengthened the concept of neofunctionalization. We also identified C-Ph1 gene orthologs in other plant species including maize, rice, barley, and Brachypodium that were closer to the 5D copy as compared to the 5A and 5B copies both in terms of sequence as well as the relative distance between the motifs thus suggesting it to be the conserved and perhaps the ancestral version of the gene. Stable RNAi silencing of the gene in Arabidopsis also showed multivalents and centromere clustering suggesting that the gene may be performing a function in diploids similar to that observed in wheat. The UTRs and promoter sequences of 5A, 5B and 5D copies were cloned directly from the genomic DNA of CS using gene specific primers. The length of the region upstream of the translational start codon was 1649bp for 5A, 934bp for 5B and 2552bp for the 5D copy. The upstream sequences al the three copies were compared with each other to gain further insight into the factors responsible for the differential expression pattern of the gene copies. The three sequences showed high sequence conservation in the proximal region of 570bp suggesting it to be the core promoter region regulating the prophase-I to metaphase-I specific expression pattern of the C-Ph1 gene homoeologs. We have also identified several copy specific-insertions/deletions and cis-acting elements that might be regulating the specific expression pattern of these copies. Additionally, several common regulatory elements were also identified that perhaps might be contributing towards the similar expression trend of the C-Ph1 gene homoeologs in different meiotic stages. We have for the first time described a novel way of delimiting the core promoter region in hexaploid wheat. Detailed bioinformatics analyses revealed that one of the variants of 5B copy proteins is a specialized motor protein involved in Holliday junction branch migration and other similar processes (unpublished results). With only 8-7% protein sequence similarity, the 3D structure of this 5B copy variant showed 100% similarity exclusively with motor proteins specific for recombination and normal cell division in bacteria, humans, drosophila and mice. The highest structural similarity with any of the other meiosis-specific protein including centromere, tubulin and other motor proteins was 50% suggesting that it has an explicit function similar to the RuvA-RuvB complex (1ixs:A, B) and Spastin proteins that are specialized motor proteins. The superimposed 3D structure of 80aa protein and the RuvA-RuvB complex proteins also exhibited high structural similarity suggesting relatedness of the two proteins. Furthermore, the expression pattern of the C-Ph1-5B copy coincides with the stage where similar motor proteins are expected to be expressing, further supporting its role as a recombination-specific motor protein. Therefore, further experiments will help in elucidating the role of C-Ph1 gene as a recombination-specific motor protein and its probable evolution via the process of neo-functionalization that happened to have evolved from structural and expressional variation and/or alternate splicing. For the above-mentioned hypothesis, the 5B-specific transcript of 243bp has been cloned and expressed in the protein expression vector in BL21 cells. The expressed protein has been extracted and purified for further use in the gel shift assay to study its association with the Holliday junction and RuvA-RuvB protein complex.

    Publications

    • Type: Journal Articles Status: Submitted Year Published: 2015 Citation: Reynolds MP, Quilligan E1, Aggarwal PK, Cavalieri A, Chapman SC, Chapotin SM, Datta SK, Gill KS, Jagadish KSV, Koehler AK, Kosina P1, Lafitte R, Mahala RS, Paterson AH, Prasanna BM, Rosegrant MW, Sharma I, Sivasankar S, Vadez V, Valluru RI, Vara Prasad PV, Yadav OP 2016. Maintaining cereal productivity under climate change through international collaboration. Global Food Security (Submitted).
    • Type: Journal Articles Status: Submitted Year Published: 2015 Citation: Shalini Pareek, Aditya Bhushan Pant, Ed Exterberria, Kanwarpal Singh Dhugga, Kulvinder Singh Gill, Sangarsh Kumar Tripathi, Gursharn Singh Randhawa 2015. Nhx1, Kup,V-atpase and Kea3 Genes are Upregulated in Salt Tolerant Accessions of Guar. Biologia Plantarum (submitted).
    • Type: Journal Articles Status: Published Year Published: 2015 Citation: Simerjeet Kaur, Kanwarpal S Dhugga, Kulvinder Gill, Jaswinder Singh 2015. Comparative analysis of cellulose synthase (CESA) genes reveals novel structural and functional conservation in hexaploid wheat. PLOS ONE 11 (1)
    • Type: Journal Articles Status: Published Year Published: 2015 Citation: AK Dhaliwal, A Mohan, G Sidhu, R Maqbool, and KS Gill 2015. An Ethylmethane Sulfonate Mutant Resource in Pre-Green Revolution Hexaploid Wheat. PLOS ONE 10 (12), e0145227.
    • Type: Journal Articles Status: Published Year Published: 2015 Citation: Gaganjot Sidhu, Amita Mohan, Ping Zhang, Amandeep Dhaliwal, Dorris Main, and Kulvinder S. Gill 2015. Sequencing-based high throughput mutation detection in bread wheat. BMC Genomics 16: 962. doi:10.1186/s12864-015-2112-1
    • Type: Journal Articles Status: Published Year Published: 2015 Citation: Christopher Navarro, Jerott Moore, Alina Ott, Eric Baumert, Amita Mohan, Kulvinder S. Gill and Devinder Sandhu 2015. Evolutionary, Comparative and Functional Analyses of the Brassinosteroid Receptor Protein gene, Bri1, in Wheat and its Relation to Other Plant Genomes. PLOS ONE (http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0127544) DOI: 10.1371/journal.pone.0127544.
    • Type: Journal Articles Status: Submitted Year Published: 2015 Citation: Ritu Batra, Gautam Saripalli, Amita Mohan, Kulvinder Singh Gill, Harindra Singh Balyan, and Pushpendra Kumar Gupta 2016. Comparative structural and functional analysis of AGPase in monocots and dicots. Functional and Integrative Genomics (Submitted).
    • Type: Journal Articles Status: Submitted Year Published: 2015 Citation: Jasdeep S. Mutti and Kulvinder S. Gill 2015. Evolution of Gene Expression Balance among Homoeologues of Natural Polyploids. New Phytologist (Submitted).
    • Type: Theses/Dissertations Status: Published Year Published: 2015 Citation: UNDERSTANDING AND UTILIZING CHROMOSOMAL PAIRING CONTROL IN POLYPLOID PLANTS, by Ramanjot Kaur Bhullar
    • Type: Theses/Dissertations Status: Published Year Published: 2015 Citation: MOLECULAR CHARACTERIZATION OF TILLER NUMBER TRAIT IN WHEAT (Triticum aestivum L. em. Thell.), By RIZWANA MAQBOOL
    • Type: Theses/Dissertations Status: Published Year Published: 2015 Citation: MOLECULAR CHARACTERIZATION OF A WHEAT ORTHOLOG OF ABCB1 (TaABCB1) GENE INVOLVED IN DWARFISM VIA INTERRUPTED AUXIN TRANSPORT, By AMANDEEP KAUR DHALIWAL
    • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: "Structure and Function of Ph1 (Pairing homoeologous 1) Gene of Wheat". Bhullar R., K.Singh, R.Nagarajan, A.Mohan, K.Gill
    • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: "Neofunctionalization of homoeologs explains multiple mechanisms of chromosome pairing control associated with the Ph1 gene of polyploid wheat.". Gill K., R.Bhullar, K.Singh, R.Nagarajan, A.Mohan
    • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: "Developing heat tolerant and climate resilient wheat". Gill K., A.Mohan
    • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: "Screening of diverse wheat germplasm for different components of heat tolerance". Alfaro T.D., Q.Graham, K.Singh, A.Mohan, N.Kumar, K.Gill
    • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: "Short-term Heat Stress During Germination and Seedling Development and its Effect on Wheat". Sharma A., S.Bakshi, J.Singh, A.Mohan, K.Gill
    • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: "Characterization of known reduced height mutants in wheat". Grant N., A.Mohan, W.F.Schillinger, K.Gill
    • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: "Neofunctionalization of homeologs explains multiple mechanism of Ph1 gene action in polyploid wheat". Singh K., R.Bhullar, A.Mohan, R.Nagarajan, K.Gill
    • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: "Difference in wheat coleoptile growth pattern and its relationship with seedling emergence". Mohan A., J.Lynch, W.F.Schillinger, K.Gill
    • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: "A Mutant Resource in Pre-Green Revolution Hexaploid Wheat". Dhaliwal A., A.Mohan, G.Sidhu, K.Gill
    • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: "Discovery of the Ph1 (Pairing homoeologous 1) gene: Light at the end of the tunnel". Singh K., R.Nagarajan, A.Mohan, K.Gill
    • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: "Understanding genetic control of coleoptile length and emergence from deep planting depths". Elbudony K.A., A.Mohan, W.F.Schillinger, K.Gill
    • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: "Improving seedling emergence of winter wheat from deep planting depths". Mohan A., W.F.Schillinger, K.Gill, S.E.Schofstoll, J.Jacobsen
    • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: "Validating dehydration screening methodology to correlate the drought impact on spring wheat to the dynamic response of proxisome proliferation.". Sanad M., A.Smertenko, P.Steel, K.Gill, K.Campbell