15-IWYP Using Next Generation Genetic Approaches to Exploit Phenotypic Variation in Photosynthetic Efficiency to Increase Wheat Yield

Lead Research Organisation: Earlham Institute
Department Name: Research Faculty

Abstract

Not required

Technical Summary

Phenotyping photosynthetic characters from diverse lines of wheat will be combined with next generation genetic approaches to enable the identification of markers and genes associated with each trait. Such knowledge will enable combinations of these traits to be rapidly incorporated into elite wheat lines to increase yields based on improved photosynthetic efficiency. Moreover, identifying the genes and mutations responsible for the traits will provide an understanding of the biology underpinning the trait and the ability to use precision genome engineering tools in the future. The project will identify wheat material, develop markers and build bioinformatics tools. All of this will be made available to the international community via CIMMYT and iPlant. The project builds upon high throughput methods and knowledge developed by the wheat yield consortium and utilises exome capture technology to discover the relevant genetic information in a cost effective manner. The project combines the diverse expertise in photosynthesis, genetics, wheat physiology and breeding from Lancaster, Liverpool, ANU and CIMMYT and leverages off related existing research.

Planned Impact

Not required
 
Description EARLHAM INSTITUTE Anthony Hall, Neil Hall, Ryan Joynson, Laura-Jayne Gardiner
Summary
? Hiring of project PDR in May 2016
? Sample collection from and genotyping of >700 CIMMYT wheat lines and 104 Watkins landrace collection members using SNP arrays/genetic capture strategies.
? Lab moved from the University of Liverpool to the Earlham institute, Norwich.
? Bisulphite sequencing and epigenetic variation analysis carried out on 104 Watkins landrace collection members.
? Population structure and relatedness analysis of CIMMYT wheat lines to aid selection of a "phenotypic tails panel" for fine phenotyping/bulk segregation analysis in years 2/3.
? Successful GWA study associating CIMMYT line derived SNPs to yield and photosynthetic efficiency related traits.
? New DNA capture based 12 Mbp genotyping platform designed and tested using the CIMMYT HiBAP panel
? New 508 Mbp genic and 277 Mbp promoter captures designed and tested using CIMMYT germplasm

Genotyping: Genotyping for 5 panels (Bread wheat diversity panel, High biomass association panel, Padmaps, Ozrip and Synthetic panel) comprising a total of 725 lines was carried out using the 35k SNP wheat breeders array (Affymetrix). DNA Samples were taken in March 2016 in CIMMYT, Obregon, from plants grown for field level phenotyping trials. The resulting SNPs were used for a Genome wide association study incorporating phenotyping data produced at CIMMYT over the growing seasons of 2015-16 and 2016-17. 104 lines identified as the core set of the Watkins land race collection were also genotyped/epityped using a 12Mbp sequence capture designed by Laura Gardiner (University of Liverpool/Earlham Institute), half of the captured DNA was sequenced directly and half was subjected to bisulphite treatment for DNA methylation analysis. Both libraries were then sequenced using a HiSeq2500 and variants were called on the resulting sequences. This produced >300k single methylation polymorphism (SMP) sites and ~600K SNP sites per line that can be used in conjunction with phenotypic measurements made at Lancaster University for future phenotype:geno/epitype association studies in year 2/3.

This 12Mbp capture was adapted by Ryan Joynson to incorporate legacy from the 35K axiom array along with tiling across genes associated with photosynthesis and biomass accumulation. Tiled genes were selected based on contributions from experts from multiple IWYP projects. The updated 12Mbp capture has been used to genotype the HiBAP panel and the members of the bread wheat panel that make up the PS-tails respiration panel. The output of this is an ultra-high-density set of SNPs for the panels that will facilitate a more high resolution GWA study along with allele mining of full genes and promoter regions of genes related to photosynthesis and biomass accumulation. This work yielded on average ~800,000 homozygous SNPs per individual along side extensive information on insertions/deletions as well as transposible element copy number.

Genome Wide Association Study: A genome wide association study is currently being carried out at the Earlham institute using the SNP makers produced for the CIMMYT Bread diversity and HiBAP panels. >40 phenotype measurements provided by the CIMMYT team will be used in this study, with the vast majority relating to yield and photosynthetic efficiency related traits. Preliminary results indicate that we can successfully identify QTLs for multiple traits related to both yield and photosynthetic efficiency. Concurrently, we are also anchoring SNP loci in the genotyping array to the newest wheat references, which will in turn increase the accuracy of identification of possible causative genes within the vicinity of the identified QTLs.

In October 2017 GWA was carried out for the HiBAP panel using phenotypic data for 2-4 reps per trait per growing season for 2 years. This study reinforced our preliminary 1 year study and yielded MTAs for multiple traits including Harvest Index, Biomass, RUE, RARSa, RARSb, Nmass/area, canopy temperature and NDWI. For this study the 35K Wheat breeders array SNPs were anchored to the Refseq1.0 reference genome by Ryan Joynson and Luzie Wingen (IWYP25), a resource which will be shared via Germinate. The outcome of this study has resulted in a publication in Plant Biotechnology Journal (IF 6.3) related to agronomic, yield, biomass and RUE related traits.

Genome Wide Association Study- Ultra-high-density SNPs:
To leverage the increase in resolution provided by the DNA capture based genotyping we are currently carrying out a further GWA study using hyperspectral reflectance indices for multiple pigmentation/water content related traits as well as the predictions modeled for VcMAX of rubisco and J MAX value relating to the maximum rate of regeneration RUBP, two of the main limiting factors in C3 photosynthesis.


Cultivar relatedness/new panel assembly: The genotyping data for the CIMMYT panels was used to assess genetic relatedness between lines. This information was used in conjunction with phenotypic measurements for yield and photosynthetic efficiency to select a genetically diverse "phenotypic tails panel" that includes panel members with high and low (relative to one another) values for the traits of interest which will be grown at the beginning of year 2 for fine phenotyping in CIMMYT and in year 3 in Lancaster. This panel will be used for bulk segregation analysis using a new capture targeting the entire known genetic space of wheat in year 3. Alongside this, during biochemical assessment of rubisco at Lancaster University, leaf samples were taken at the same time as these measurements which will be the subject of a transcriptomic study assessing the differential expression profiles between lines showing the highest and lowest measurements for various biochemical tests relating to photosynthetic rate. This will be completed by the end of year 3.
Exploitation Route Material generated by this project is already feeding into the CIMMYT prebreeding programme. Markers are now being tested for use in breeding programme.
Sectors Agriculture, Food and Drink
URL https://iwyp.org/
 
Description Markers developed are now being tested for integration into the CIMMYT breeding programme. The material identified has gone forward into pre-breeding crosses. The capture platform developed is now being sold commercially by ROCHE. We have now used the genotype data to identify 3 exotic wheat alleles associated with heat resistance. These markers are now being used by CIMMYT and two international breeding companies
Sector Agriculture, Food and Drink
 
Description Big data and agriculture in India
Geographic Reach Asia 
Policy Influence Type Participation in a guidance/advisory committee
 
Description 18-BTT: A PATHWAY TO THE EXPLOITATION OF EPIGENETIC VARIATION IN UK, US AND INTERNATIONAL BREEDING PROGRAMMES
Amount £273,500 (GBP)
Funding ID BB/S020942/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2019 
End 03/2021
 
Description Exploiting night-time traits to improve wheat yield and water use efficiency in the warming climate of North-western Mexico
Amount £541,034 (GBP)
Funding ID BB/S012834/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2019 
End 05/2022
 
Description SEQUENCING THE GENIC PORTION OF SEEDS OF DISCOVERY ADVANCE PRE- BREEDING GERMPLASM TO UNCOVER THE GENETIC VARIATION
Amount £330,000 (GBP)
Funding ID BBS/OS/NW/000017 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2016 
End 10/2019
 
Title promoter exon capture platform 
Description Whole genome shotgun re-sequencing of wheat is expensive because of its large, repetitive genome. Moreover, sequence data can fail to map uniquely to the reference genome making it difficult to unambiguously assign variation. Re-sequencing using target capture enables sequencing of large numbers of individuals at high coverage to reliably identify variants associated with important agronomic traits. Previous studies have implemented cDNA/exon or gene-based probe sets where promoter and intron sequence is largely missing alongside newly characterized genes from the recent improved reference sequences. Results We present and validate two gold standard capture probe sets for hexaploid bread wheat, a gene and a putative promoter capture, which are designed using recently developed genome sequence and annotation resources. The captures can be combined or used independently. We demonstrate that the capture probe sets effectively enrich the high confidence genes and putative promoter regions that were identified in the genome alongside a large proportion of the low confidence genes and associated promoters. Finally, we demonstrate successful sample multiplexing that allows generation of adequate sequence coverage for SNP calling while significantly reducing cost per sample for gene and putative promoter capture. Conclusions We show that a capture design employing an 'island strategy' can enable analysis of the large gene/putative promoter space of wheat with only 2x160 Mb probe sets. Furthermore, these assays extend the regions of the wheat genome that are amenable to analyses beyond its exome, providing tools for detailed characterization of these regulatory regions in large populations. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? Yes  
Impact Currently being used by DFW, CIMMYT and US CAPS projects 
 
Title Genotyping of 150 CIMMYT wheat lines (paper DOI 
Description 35K affymetrix array data for the High Biomass Association Panel of 150 spring wheat lines from CIMMYT. A paper explaining the panel and the use we made of these snps is available here: http://dx.doi.org/10.1111/pbi.13052 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact Facilitated the identification of multiple marker trait associations which have now been incorporated into CIMMYT spring wheat marker assisted selection program. 
 
Title Watkins core collection re-sequencing data 
Description Re-sequence data for the Watkins collection 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact International collabration 
URL https://grassroots.tools/data/under_license/toronto/
 
Title promoter exon capture data 
Description The data contain the design space for the promoter and exon used in our capture 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact Whole genome shotgun re-sequencing of wheat is expensive because of its large, repetitive genome. Moreover, sequence data can fail to map uniquely to the reference genome making it difficult to unambiguously assign variation. Re-sequencing using target capture enables sequencing of large numbers of individuals at high coverage to reliably identify variants associated with important agronomic traits. We present two gold standard capture probe sets for hexaploid bread wheat, a gene and a promoter capture, which are designed using recently developed genome sequence and annotation resources. The captures can be combined or used independently. The capture probe sets effectively enrich the high confidence genes and promoters that were identified in the genome alongside a large proportion of the low confidence genes and promoters. We use a capture design employing an 'island strategy' to enable analysis of the large gene/promoter space of wheat with only 2x160 Mb NimbelGen probe sets. Furthermore, these assays extend the regions of the wheat genome that are amenable to analyses beyond its exome, providing tools for detailed characterization of these regulatory regions in large populations. Here, we release the targeted sequence of the capture probe sets on the wheat RefSeqv1, the design space that was used to tile our capture probes across and finally the positions of the probes themselves across this design space for both the gene and promoter capture probe sets. 
URL https://opendata.earlham.ac.uk/wheat/under_license/toronto/Gardiner_2018-07-04_Wheat-gene-promoter-c...
 
Description CIMMYT seeds of discovery 
Organisation Australian National University (ANU)
Country Australia 
Sector Academic/University 
PI Contribution Collaboration to provide Advance genetic approaches
Collaborator Contribution Access to wheat material and phenotyping data
Impact None yet
Start Year 2016
 
Description CIMMYT seeds of discovery 
Organisation International Centre for Maize and Wheat Improvement (CIMMYT)
Country Mexico 
Sector Charity/Non Profit 
PI Contribution Collaboration to provide Advance genetic approaches
Collaborator Contribution Access to wheat material and phenotyping data
Impact None yet
Start Year 2016
 
Description IWYP 
Organisation Australian National University (ANU)
Department Division of Plant Sciences
Country Australia 
Sector Academic/University 
PI Contribution My group lead this work, we are involved in the genotyping and bioinformatics
Collaborator Contribution The three other groups are involved in generating the field phenotyping and plant physiology measurements.
Impact none yet
Start Year 2016
 
Description IWYP 
Organisation International Centre for Maize and Wheat Improvement (CIMMYT)
Country Mexico 
Sector Charity/Non Profit 
PI Contribution My group lead this work, we are involved in the genotyping and bioinformatics
Collaborator Contribution The three other groups are involved in generating the field phenotyping and plant physiology measurements.
Impact none yet
Start Year 2016
 
Description IWYP 
Organisation Lancaster University
Department Lancaster Environment Centre
Country United Kingdom 
Sector Academic/University 
PI Contribution My group lead this work, we are involved in the genotyping and bioinformatics
Collaborator Contribution The three other groups are involved in generating the field phenotyping and plant physiology measurements.
Impact none yet
Start Year 2016
 
Description KWS 
Organisation KWS UK
Country United Kingdom 
Sector Private 
PI Contribution Generated double haploid population for the IWYP project and provide material for INTREPID and the BBSRC/EAGER work
Collaborator Contribution Know of the techniques and approaches we are using, early access to the data we generate
Impact Double haploid seed population
Start Year 2017
 
Description RAGT 
Organisation RAGT Seeds
Country United Kingdom 
Sector Private 
PI Contribution Identify key material for our EAGER project and Design future wheat. Support for collaboration with IBM.
Collaborator Contribution Discussion of methods and analysis of material relevant to RAGT.
Impact none yet
Start Year 2018
 
Description 10 plus wheat genome project workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The meeting was to provide a consortium update on the 10 plus wheat genome project
Year(s) Of Engagement Activity 2018
URL http://www.10wheatgenomes.com
 
Description Agro-Biodiverstiy conference -India 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited speaker at "Present and Future Opportunities for Using Biodiversity for Wheat Improvement"
Year(s) Of Engagement Activity 2016
 
Description Hosting a PhD student from CIMMYT (Mexico) for training 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact The Hall lab group hosted a Phd student supervised by Dr Matthew Reynolds (CIMMYT) and Dr John Faulkes (University of Nottingham) for training in crop genetics.

Over a period of 3 days we introduced her to concepts surrounding phenotype - genotype association analysis culminating in the successful application of GWAS to her data.

This lead to a very well recieved poster presentation at the Plant and Animal Genomes conference in 2019:

https://pag.confex.com/pag/xxvii/meetingapp.cgi/Paper/33006

Strategies for achieving genetic gains in yield potential must combine enhanced above-ground dry matter as well partitioning to the grain (harvest index). The present study aligned with NIFA-IWYP (International Wheat Yield Partnership) project N-IWYP700* aimed to identify grain partitioning traits that help maximize grain yield in high biomass backgrounds.
A High Biomass Association Panel (HiBAP) comprised of 150 spring wheat elite genotypes was phenotyped in 2015-16 and 2016-17 at CIMMYT experimental station Norman E. Borlaug located in NW Mexico. Physiological traits measured included biomass, plant length (height, spike, awns, peduncle, internode 2 & 3), organ DM partitioning (spike, leaf lamina, true stem and leaf sheath) and fruiting efficiency (# of grain set per unit spike dry weight) at anthesis (GS65) + 7 days and at harvest, biomass, grain yield, yield components and harvest index was measured. BLUEs (Best Linear Unbiased Estimators) from the cross-year analysis and molecular markers generated using the 35K Wheat Breeders Axiom array were used to carry out a genome-wide association study (GWAS).

Novel marker-trait associations for the grain partitioning traits were identified mainly in chromosomes 5B and 6A; resulting in 13 at anthesis (e.g. LamPI, spike length) and 9 at harvest (e.g. HI, FE_GSP). More detailed results will be presented and their potential application in breeding programs in marker-assisted selection discussed.
Year(s) Of Engagement Activity 2018
URL https://pag.confex.com/pag/xxvii/meetingapp.cgi/Paper/33006
 
Description Monogram Grain Conference 2019 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Poster presentation of GWAS of 150 spring wheat lines associating agronomic, yield and biomass related traits to SNP markers for use in the CIMMYT (Mexico) pre-preeding marker trait assisted breeding program. This work is now published here: https://onlinelibrary.wiley.com/doi/full/10.1111/pbi.13052 and was carried out as part of the International Wheat Yield Partnership to provide essential marker trait associations to wheat breeders at CIMMT for tracking high biomass and radiation use efficiency, both which are thought to be a current bottleneck in genetic gains in their populations.

The poster was well received and gained a lot of attention during the poster session. A link made during this yielded an on going collaboration with scientists at Rothamsted Research (Mathew Paul).
Year(s) Of Engagement Activity 2018
URL http://www.monogram.ac.uk/MgNW2018.php
 
Description Oral Presentation The Rank Prize meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The meeting was run as an invitational meeting run by the "The Rank Prize Fund" and was named "The shape of wheat to come". The meeting was attended by 30 early career scientists and around 20 more seasoned scientists with the aim of introduction of early career scientists to long established members of the wheat community (including breeding companies). A participant was invited from the most influential groups studying wheat mainly from the UK and France. Each participant was required to
A brief description below:

The Trustees' Advisory Committee on Nutrition is arranging a series of symposia on topics that are of current interest. In 2018 we intend to organise a mini-symposium on The Shape of Wheat to Come which will be held at The Wordsworth Hotel, Grasmere, Cumbria, England, from 19th to 22nd March 2018.

The aim of the meeting is to provide a forum in which leading scientists and young research workers can meet and interact, in order to stimulate discussion and to advance the development of the subject. Attendance is by invitation only and will be limited to about 30 participants. The main speakers will have about 40 minutes for their talk and related discussion and the young scientists will be allocated 20 minutes to give an account of their work. The value of these symposia lies in their small, fully interactive nature, not only during the presentations and discussion, but at other times over meals, in the hotel bar and during recreational periods.
Year(s) Of Engagement Activity 2018
 
Description Plant and Animal genomes conference 2019 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Attendance to plant and animal genomes conference 2019- including 2 x poster presentations. One poster contained work carried out and reported in a recent publication ( 10.1111/pbi.13052). This poster recieved a lot of attention and we were approached multiple times by scientists working in wheat barley and rice along with making contact with a member of the breeding community (KWS) who was interested in the marker trait associations (MTAs) we had discovered and presented in the poster. The second poster featured collaborative work we have carried out with Dr John Faulkes from the University of Nottingham, which also gain wide interest as it denotes the first identifiaction of markers relating to various stem and spike partitioning indices.

These presentations also have lead to further discussions with scientists from NIAB about the use of a capture probe set we have designed as part of Designing Future Wheat ISP and our International Wheat Yield partnership grant.
Year(s) Of Engagement Activity 2019
URL https://www.intlpag.org/2019/
 
Description Talk and discussion with Elsoms seeds 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Industry/Business
Results and Impact The talk was to breeders and crop scientist work at Elsoms seed, The aim was to raise awareness of the work that Design future wheat and my group were doing. This lead to a letter of support for our BBSRC grant and a line of communication with their wheat and brassica breeders.
Year(s) Of Engagement Activity 2018
 
Description Training course delivery: BecA-ILRI Hub 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Myself and Laura Jayne Gardiner were approached to deliver a 1 week training session at the BecA-ILRI Hub, Nairobi, Kenya. The course was a 3 month training program ran by the Alliance for Accelerated Crop Improvement in Africa. Our contribution to the program was training in the background of NGS technologies and their use in crop development along with teaching applied bioinformatics fundamentals to the group of students. The students on the course were from >15 countries in Africa and returned to their host institutes with a fantastic base knowledge of bioinformatics which they will apply for crop/livestock improvement throughout Africa.
Year(s) Of Engagement Activity 2018
URL https://acaciaafrica.org/bioinformatics-community-practice/members-and-management/