Genomics-assisted selection of Solanum chilense introgression lines for enhancing drought resistance in tomatoes

Lead Research Organisation: Cranfield University
Department Name: School of Water, Energy and Environment

Abstract

Tomato, the biggest food crop after cereals and potato, is a vital source of nutrients, vitamins and other health promoting metabolites in the human diet: production must increase, and breeding for sustainable production is a high priority. Breeding for improved water use efficiency and ability to give high yields where water availability is poor are important traits for tomato, and other Solanaceous crops, including potato and Brinjal. Solanum pennellii, Solanum chilense and Solanum sitiens are the three tomato wild relatives that are found in arid areas and have the greatest adaptation to water-limited environments, and each has a distinct morphology, classification and ecotype. However, good genetic and genomic resources only exist for S. pennellii, where well-established and highly successful strategies have been employed to exploit allelic variation.
In this project we will develop novel genetic and genomic resources for the remaining drought-adapted species, S. chilense and S. sitiens. We will generate >400 x depth genomic sequence data and perform de novo genome assembly for these two genomes to provide an excellent reference sequence complete with structural variants at different scales; this will be done in close collaboration with European bioinformatics experts working with other Solanum genomes (Wageningen UR, RWTH Aachen University and Max Planck Institute of Molecular Plant Physiology), and will provide strong capacity building in bioinformatic skills and computing power.
Genome sequences will be used for SNP marker design and a genetic map constructed for an S. chilense x S. lycopersicum population. Genome sequences will become extremely valuable resources as QTL studies progress to the gene level.
We will rapidly create, using a high-throughput SNP genotyping platform, a population of introgression lines for S. chilense starting with a unique hybrid obtained by IIVR, now in the BC1F1 generation. The completed library will be screened for water use efficiency, root traits and resistance to wilting to discover new QTL. A rapid backcross program in MicroTom will be used to phenotypically select introgressions arising from a complex hybrid containing alleles from two S. chilense and one S. peruvianum accession. Introgressions present at the end of the selection process will be precisely defined by genome re-sequenced in preparation for fine mapping.

Technical Summary

There are three wild relatives of cultivated tomato (Solanum lycopersicum) that are well known to be adapted to the very arid regions of South America: S. pennellii, S. chilense and S. sitiens. Much progress has been made in exploiting the germplasm of S. pennellii, which easily forms hybrids with S. lycopersicum. However, S. chilense and S. sitiens are more problematic as they fall outside the "esculentum group" for cross compatibility, but they remain a rich source of valuable alleles. In this project, we will develop the genomic resources for both S. chilense and S. sitiens by performing de novo genome assembly, and will develop introgressed line (IL) and inbred backcross line (IBL) populations to capture the S. chilense genome for QTL mapping and exploitation in breeding programs. This will utilise a rare hybrid between S. chilense and S. lycopersicum which has now been progressed to BC1F1. We will use the ILs to discover novel alleles for root traits and for water use efficiency, key target traits to improve sustainable water use in tomato production. Genomes will be sequenced to a depth of >400X using mate pair and paired end libraries with a range of insert sizes up to 40 kbp and assembled de novo with assistance from experienced project partners. OpGen Argus will be used for optical mapping to join scaffolds and improve the quality of the draft genomes. Genome re-sequencing of parental lines will be used to develop a set of 384 SNPs which will be used to select IL and IBL populations. A complex bridging line containing DNA from S. chilense and S. peruvianum which hybridises with cultivated tomato will be re-sequenced, and used to capture introgressions that confer wilting resistance by backcrossing five times to model tomato cultivar MicroTom with recurrent phenotypic selection. Re-sequencing will delimit captured introgressions.
Physiological mechanisms will be investigated in detail for introgression lines that show improved WUE or wilt resistance.

Planned Impact

The genome sequences and genetic resources developed in this project and the QTLs identified will lead directly to the development of novel tomato varieties that have a reduced requirement for irrigation. Populations will be created using a current Indian cultivar as genetic background; therefore beneficial introgressions could potentially be delivered very rapidly by the Indian partners without extensive further breeding and selections.
Reduction in irrigation is beneficial to the environment as it will contribute to a more sustainable use of water resources which are currently often used in an unsustainable manner resulting in depletion of ground waters and reduced river flows, particularly in regions such as South East Asia, Southern Europe and California. Since the UK relies heavily upon imports of both fresh market and processing tomatoes, the sustainability of production overseas is important for UK food security. Improved water use efficiency can also increase yields and farmer incomes where availability of water is limiting to production: cultivars with higher water use efficiency will produce a higher yield for the same water inputs.
In the longer term, the same genetic and genomic resources can be used to breed tomato cultivars with resistance to a range of pest and diseases which will in turn increase profits and reduce the use of chemical pesticides. Higher yields and lower prices resulting form crop improvement in tomato will increase consumption and contribute to a healthier diet. A range of other cultivar improvements are possible when S. chilense and S. sitiens introgression lines become available to tomato breeders. The genome sequences will facilitate rapid marker assisted selection by providing hundreds of thousands of polymorphisms for molecular breeders to use.
The results of this project will allow UK and India-based breeding companies to develop novel cultivars which will increase sales of profitable hybrid seeds, and improve global market share. QTL discovered in tomato can potential by used in other Solanum crops through allele surveys in parallel germplasm collections, e.g. in potato, pepper and brinjal.
The project will increase UK and India's capability in advanced bioinformatics for researchers working with horticultural crops who will develop the skills and computing facilities for analysing very large data sets arising from next generation sequencing platforms; linking this capability to existing graduate teaching programs at Cranfield will cascade this skill to up to 20 MSc students each year who go on to work in a wide range of bioscience contexts.

Publications

10 25 50
 
Description We have produced genome assemblies for the two wild species related to tomato: Solanum chilense and Solanum sitiens. Assemblies were constructed with Illumina paired-end data, PacBio Sequel data, 10x Genomics Chromium and optical mapping and provide a resource for tomato breeding and genetics research. We have also obtained RNAseq data that helps in annotation of the genomes. We have created back-cross inbred line and introgression line populations as resources for understanding the key traits that are available from the wild species Solanum chilense. These populations have been genotyped using the most recent technology "genotype by sequencing" and will be ready after further development through two more generations in India. We have shown that S. chilense differs from cultivated tomato by having roots that are able to overcome barriers more easily and this may be the reason that this species is considered to be drought resistant. We have preformed backcrossing and selection from a S. arcanum/S. chilense bridging line to select drought resistant lines in the MicroTom genetics background - these have been resequenced and the Seeding Catalyst additional funding was used to identify introgressions through analysis of the sequence data, and to further phenotype the lines to establish responses to soil drying. Further mapping will then be used to identify genes that are responsible for these altered responses.
In addition we have developed several new bioinformatics tools and approaches: a tool (MapOptics) to improve analysis of optical mapping data (published), a new method of phasing heterozygous genomes (paper in preparation), and a new concept for genome version control to improve data management practices when updating reference genomes (follow-up research grant submitted).
Exploitation Route The genomes assemblies are being made public on the Sol Genomics Network initially, in advance of publication. Genetic materials will be made available by request. Genetic and genomic resources will help others to breed tomato cultivars with improved characteristics such as drought resistance and virus resistance.
Sectors Agriculture, Food and Drink

 
Description Cranfield Industrial Partnership Studentship
Amount £91,410 (GBP)
Organisation Rijk Zwaan B.V. 
Sector Private
Country Netherlands
Start 02/2017 
End 01/2020
 
Description Direct Funding
Amount £39,705 (GBP)
Organisation Rijk Zwaan B.V. 
Sector Private
Country Netherlands
Start 09/2017 
End 09/2018
 
Description Seeding Catalyst Award
Amount £24,486 (GBP)
Funding ID WXA6082N 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2018 
End 02/2019
 
Title MapOptics 
Description Bionano optical mapping is a technology that can assist in the final stages of genome assembly by lengthening and ordering scaffolds in a draft assembly by aligning the assembly to a genomic map. However, currently, tools for visualization are limited to use on a Windows operating system or are developed initially for visualizing large-scale structural variation. MapOptics is a lightweight cross-platform tool that enables the user to visualize and interact with the alignment of Bionano optical mapping data and can be used for in depth exploration of hybrid scaffolding alignments. It provides a fast, simple alternative to the large optical mapping analysis programs currently available for this area of research. 
Type Of Material Data analysis technique 
Year Produced 2018 
Provided To Others? Yes  
Impact n/a 
URL https://github.com/FadyMohareb/mapoptics
 
Title Solanum sitiens and Solanum chilense genome assemblies 
Description Genome assembly data in form of sequence reads, contigs and scaffolds is being made available on the Sol Genomics Network in advance of publication. Transcriptome data is also available for release for purposes of gene annotation. 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? Yes  
Impact n/a 
URL https://solgenomics.net/
 
Description Cranfield collaboration with Indian Institute of Vegetable Research, Varanasi 
Organisation Indian Institute of Vegetable Research
PI Contribution We are engaged in a joint BBSRC-DBT funded project "ChilBix" and we provide expertise in tomato genetics and bioinformatics.
Collaborator Contribution Tomato breeding and genetics. Provision of germplasm, population development and collaborative work on genotyping and genome assembly.
Impact Draft genome assemblies for tomato wild species. Populations for QTL mapping in tomato.
Start Year 2015
 
Description BBSRC Seeding Catalyst Award Event 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Results of seeding catalyst projects were presented to industry representatives (growers, consultants, seed companies, academics), and the CHAP and AgriEPI AgriTech centre facilities were discussed and a tour took place. A discussion was had about how to engage with use of facilities and with the research activities presented.
Year(s) Of Engagement Activity 2019
 
Description Plant and Animal Genomes Conference 2016, San Diego 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Poster presented by Tom Kurowski, Bioinformatics Research Assistant.
Title of poster: Development of genomic resources for Solanum chilense and Solanum sitiens.
Authors: Kurowski T, Almeida, P, Mohareb, F, Niranjan S, Singh D, Geetika B, Sarvesh P, Prasanna HC and Thompson AJ.

Discussions at the conference influenced the future strategy of the genomics research at Cranfield University and its partners in the Indian Institute of Vegetable Research.
Year(s) Of Engagement Activity 2016
 
Description Presentations at Baranas Hindu University, Institute of Agricultural Sciences, Varanasi 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Prof Andrew Thompson and Dr Fady Mohareb from Cranfield University visited Institute of Agricultural Sciences, Baranas Hindu University, Varanasi, India. Each gave a presentation to the academic staff and postgraduate and undergraduate students. We also visited the research facilities and had a round-table discussion with senior academic staff. Titles of presentations: Andrew Thompson: Genetic control of water use efficiency and drought resistance in horticultural crops Fady Mohareb: Next generation sequencing to unravel the genetic basis of key plant genomes: case studies.

The presentation generated an in-depth discussion about traits required for local water-limited environments and high temperatures.
We discussed complementarity in soil lysimeter facilities and the potential for student exchanges.
The renewal of a Memorandum of Understanding between Cranfield University and Baranas Hindu University was discussed.
Year(s) Of Engagement Activity 2017
 
Description Solanaceae Genomics 2015 Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Oral presentation by postdoc Pedro Almeida representing the BBSRC project entitled: Genomics-assisted selection of Solanum chilense introgression lines for enhancing drought resistance in tomatoes. The presentation was in the Solanaceae Genomics 2015 meeting in Bordeaux, France.

Presentation stimulated discussion making comparisons with Solanaceae sequencing projects going on internationally.
Year(s) Of Engagement Activity 2015
 
Description Solanaceae Genomics 2016 Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Poster presentation at a conference that included academic and industry delegates. Title: "Exploiting genetic variation to improve water use efficiency and drought resistance in tomato"
Authors: Andrew Thompson, Tomasz Kurowski, Pedro Almeida, H.C. Prasanna, Niranjan Shirgoankar, Emmanuel Solomon, Lazaro E. P. Peres and Fady Mohareb
Year(s) Of Engagement Activity 2016
 
Description Visit and presentation at the University of Hyderabad, Repository of Tomato Genomic Resources, and School of Life Sciences. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Prof Andrew Thompson and Dr Fady Mohareb from Cranfield University visited the University of Hyderabad, India hosted by Professor Ramesh Sharma who is Director of the Repository for Tomato Genomic Resources and leads a large group of researchers engaged with tomato genetics and genomics research.
Titles of presentations:
Andrew Thompson: Genetic analysis of tomato development in the post-genome era: from rootstocks to inflorescence architecture.
Fady Mohareb: Next generation sequencing to unravel the genetic basis of key plant genomes: case studies.

Future collaboration to utilise Prof Sharma's TILLING population to investigate genetic loci of common interest was arranged.
Future exchange of students was discussed.
Exchange of genetic material, including bacterial strains related to carotenoid cleavage dioxygenases was arranged.
Year(s) Of Engagement Activity 2017
 
Description Visit to the Indian Institute of Vegetable Research 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Prof Andrew Thompson and Dr Fady Mohareb from Cranfield University visited the ICAR Indian Institute of Vegetable Research, Varanasi, India and each gave a presentation to the staff and visited the field trials and met with the institute Director and staff engaged in vegetable breeding.
Titles of presentations:
Andrew Thompson: Genetic control of water use efficiency and drought resistance in horticultural crops
Fady Mohareb: Next generation sequencing to unravel the genetic basis of key plant genomes: case studies.

Progress on joint project discussed and reciprocal visit arranged.
Year(s) Of Engagement Activity 2017