The Peanut Research Foundation has completed the process of selecting an aflatoxin research project. A special call for aflatoxin related proposals ended in February and the board considered six excellent proposals. The board voted to fund Dr. Peggy Ozias-Akins’ proposal entitled “Genetic Approach to Mitigate Aflatoxin Contamination in Peanut“.
Development of genetic resources for drought and aflatoxin mitigation
Principal Investigator: Dr. Josh Clevenger, HudsonAlpha Institute, Huntsville, AL
Deploying genetic factors to mitigate pre-harvest aflatoxin contamination is the first line of defense for peanut production. For the 2019 RFP, we proposed to investigate factors that are related to seed and test traits. This work is progressing well, but the data points needed to design the next step in this research will not be available for the 2020 RFP. Instead, we propose here to continue our work identifying and developing genetic tools to mitigate pre-harvest aflatoxin contamination by developing genetic tools to select for drought tolerance. Drought tolerance contributes to low aflatoxin accumulation during late season drought conditions. A set of progenies from a cross between PI 502126 and Georgia Green were selected for drought tolerance and aflatoxin contamination over multiple years. Two progenies of that cross, named C431-1-4 and C431-1-7, showed superior drought tolerance, low aflatoxin, and yield under drought conditions. Three additional progenies show moderate drought tolerance and aflatoxin contamination. Breeding for drought tolerance has been a slow endeavor. A progeny with superior drought tolerance and yield has not yet been selected for even under well-watered conditions. Current field-based screening methods are not conducive for developing markers to select for drought tolerance because of the physical restraints of screening enough individuals. A method to screen individual seedlings would unlock the ability to effectively map new sources of drought tolerance and screen breeding populations at early stages for combining drought tolerance with superior yield and grade. With the tools developed as part of the peanut genome initiative, it is now possible to detect the alleles from the C431 cross and identify potential variation using molecular markers. This project has two main goals; to use genomic techniques to identify candidate variation in the superior drought-tolerant and low-aflatoxin accumulating lines, C431- 1-4 and C431-1-7, and to use those lines to develop a seedling-based screening method with automatic phenotyping for rapid selection of drought-tolerant individuals. The proposed deliverables are candidate markers to select for drought tolerance/low aflatoxin from a novel source and a screening method that can become a platform for rapid drought tolerance selection in breeding populations, marker-assisted breeding, and marker development for drought tolerance. The overall goal is to develop the molecular tools to incorporate low aflatoxin and drought tolerance into elite germplasm effectively.
Characterizing and pyramiding multiple disease resistances for sustainable peanut production
Principal Investigator: Dr. Peggy Ozias-Akins, Institute of Plant Breeding, Genetics, & Genomics, University of Georgia
Resistance to leaf spot and other pests/diseases in cultivated peanut can be greatly improved by accessing the diversity found in wild relatives of the crop. While direct crosses between wild diploid species and cultivated tetraploid introduce the complication of unbalanced chromosome numbers (hexaploid route), knowledge of genome affinities and generation of crosses with balanced chromosome numbers has led to a more successful strategy (tetraploid route). Using the latter route, significant progress over the last five years has been made to generate new synthetic tetraploids and create hybrids with peanut. These materials have been screened for late leaf spot, TSWV resistance, and peanut-like growth habit in the field, and some already are known to carry alleles for nematode resistance. Further characterization of progeny and backcrosses of these materials through phenotyping, genotyping, and sequencing is proposed in order to leverage the resources generated through the peanut genome and associated projects. Genotypic and phenotypic data will inform selection of lines with good agronomic characteristics and novel sources of disease resistance that can be used in cultivar development programs.
The primary objective is to incorporate pre-breeding materials generated through a NSF-sponsored project into the joint USDA-UGA peanut breeding program to capture new alleles for leaf spot and other pest/disease resistances in lines that have the morphological characteristics and productivity of cultivated peanut and are adapted to cultivation in the southeastern US.
The specific objectives are to advance disease-resistant and/or high-yielding selections from populations of four synthetic tetraploids by cultivated peanut crosses to the next generation and use a subset in additional backcrosses with cultivated peanut parents that afford the opportunity to pyramid traits. Where applicable, molecular markers for QTL will be used to expedite recovery of combinations of QTL for multiple pest resistances.
Incorporating new, wild species-derived, resistances against late leaf spot into elite peanut cultivars
Principle Investigator: Dr. David Bertioli, Institute of Plant Breeding, Genetics, & Genomics, University of Georgia
Cultivated peanut has an exceptionally narrow genetic base. This imposes limitations on the improvements that can be made by breeding with peanuts of pure pedigree, in particular with regard to pest and disease resistance. Here we propose to advance the incorporation of new wild species-derived sources of resistance to late leaf spot into agronomically elite peanut lines from the Southeast of the USA. The proposed work for 2021 builds on a successful first year in 2020 and is based on four independent sources of resistance (A. cardenasii, A. stenosperma, A. batizocoi and A. valida), three of them already incorporated in advanced lines. We propose to use a combination of field selection and the cutting-edge genetic tools that have been developed in the wake of the peanut genome project. These tools allow the identification and tracking of wild disease resistances with specific DNA-based markers. In the medium term, we expect to create new peanut cultivars adapted to the Southeastern USA, with these very strong disease resistances. This will reduce the necessity for fungicide sprays and input costs, improving profitability, reducing environmental impact.
The research and development will concentrate on disease resistances derived from four wild species: A. cardenasii (Objective 1), A. stenosperma (Objectives 2 and 3), A. batizocoi (Objective 2), and A. valida. Objectives are to:
- Combine wild-species derived late leaf spot resistance with early leaf spot resistance into elite genetic backgrounds for the Southeast of the USA. These resistances both originally derive from the wild species A. cardenasii and are both already incorporated into advanced genetic backgrounds.
- Advance and analyze late leaf spot resistance in a previously developed highly back-crossed population derived from a cross between a wild-species derived tetraploid hybrid with an elite breeding line. (The wild species being A. stenosperma and A. batizocoi.)
- Select superior genotypes from F4, BC1F3 and BC2F2 populations derived from crosses between elite peanut and a wild-species derived tetraploid hybrid, advance a generation, and perform a genetic characterization on selected genotypes. (The wild species being A. valida and A. stenosperma.)
Developing Genomic Resources for Breeding Leaf-Spot Resistant Peanut (Arachis hypogaea) Cultivars for the Virginia-Carolinas Region
Principal Investigator: Dr. Jeffery Dunne, North Carolina State University
The long-term objective of this research proposal is to develop a high-throughput genotyping pipeline specifically for the improvement of Virginia-type peanut cultivars for the Virginia-Carolinas (VC) production region. Antecedently, this requires improved marker discovery in Virginia-type peanuts and development of a genotyping approach that is both efficient and economical. A four-step approach is envisioned to accomplish this objective consisting of: 1) Development of a Virginia-type reference genome, 2) Alignment of whole-genome sequencing (WGS) data from 48 diverse genotypes to this reference genome for single nucleotide polymorphism (SNP) discovery, 3) In silico digestion of the reference genome to determine an optimal pair of restriction enzymes for genotype-by-sequencing (GBS) in order to capture the highest number of SNPs for a genome-wide association study (GWAS), and 4) A use case involving high throughput genotyping and intensive phenotyping to develop marker-trait associations in cultivated and wild species for resistance to leaf spot; a persistent, devastating fungal disease throughout the entire United States peanut producing region. Once established, this pipeline will be easily amenable to any market type or trait of interest in peanut. Furthermore, on a per sample basis, genotyping costs will be reduced ~75% compared to the only currently available alternative. This will allow a breeding program to break even on genotyping costs after 2,850 samples, which is realistically reached by a single public sector plant breeding program within four years. This proposal is being submitted in response to the request by the Peanut Research Foundation to develop high-quality markers for marker-assisted selection and will specifically target leaf spot (early and late) resistance. Research Objectives
- Develop a high-quality reference genome for Virginia-type cultivar Bailey II
- Single Nucleotide Polymorphism (SNP) discovery using Whole-Genome Sequencing (WGS) of diverse lines within the North Carolina State University (NCSU) peanut breeding program
- In silico digestion of the Bailey II reference using paired enzymes in order to design a tailored Genotyping-By-Sequencing (GBS) protocol
- Characterize leaf spot (early and late) resistant loci through image analysis of advanced breeding lines in the NCSU peanut breeding program
Proposal Title: Precision Breeding Using Molecular Genetic Tools to Develop Disease Resistant Peanut Cultivars
Principal Investigator: Dr. Corley Holbrook, USDA-ARS, Tifton, GA
We have an Arachis hypogaea breeding line that has three well-defined segments of the wild A. cardenasii chromosomes conferring excellent resistance to late leaf spot. Molecular markers associated with these chromosomal segments are also available. We have already made numerous hybridizations and are using marker assisted selection (MAS) in an accelerated backcross breeding scheme to introgress resistance to leaf spot in high yielding genetic backgrounds adapted to US peanut growing regions. We are using several recurrent parents (genotypes with and without high O/L; genotypes with and without nematode resistance; runner market-types and Virginia market-types) and expect to have several finished products in the near future. In 2019 we conducted replicated yield trials under fungicide sprayed and non-sprayed conditions with twelve selections from the first backcross generation. Twelve these lines were advanced to multi-environment yield trials in 2020 to provide data to support potential cultivar release(s). In 2020 we also conducted replicated yield trials under fungicide sprayed and non-sprayed conditions with 26 selections from the second backcross generation. We have completed the third backcrosses and grew the F1 hybrid plants in 2020. We have also completed crosses to begin MAS for resistance to white mold. Funding from the Peanut Research Foundation will allow us to expand these breeding efforts to more rapidly generate the data and the seed that will be necessary to support cultivar release(s). We anticipate releasing cultivars in two or three years. Increased funding during this time will also enable us to simultaneously increase seed using summer and winter nurseries. This will save 2-3 years in the usual time necessary for seed increase so that these cultivars will more rapidly be available to peanut farmers.
- Develop high-yielding peanut varieties with high levels of resistance to leaf spot.
- This will include cultivars with and without nematode resistance; cultivars with and without high oleic acid; and will include runner and Virginia market-types.
- Seed increase and purification using summer and winter nurseries for lines that are expected to be released as cultivars.
- Develop breeding populations and begin to use MAS for resistance to white mold.
Developing Multi-Species Introgression Lines with Prescription Marker Panels for Breeding Leaf Spot Resistant Peanut Cultivars
Principal Investigator: Dr. Ryan Andres, North Carolina State University
The ultimate objective of this research proposal is to develop germplasm lines carrying early and late leaf spot resistance genes originating from both Arachis cardenasii and A. diogoi. Based off previous research, the best performing A. cardenasii and A. diogoi introgression lines have been identified and crossed with one another to develop segregating populations. These lines and their parents will be subjected to whole-genome sequencing (WGS) to identify the introgression blocks. Single nucleotide polymorphism (SNP)-based markers will be designed that flank each introgression. These markers will then be used in a marker-assisted selection (MAS) program to develop a panel of inbred lines containing different combinations of the introgression blocks. Subsequently, these inbred lines will enter a replicated field test to determine which combinations of introgression blocks display the highest levels of resistance to leaf spot. Once identified, these lines will be released to the wider peanut research community along with the marker information needed to utilize each line in a marker-assisted cultivar development program. This should enable the rapid development of agronomically elite cultivars possessing exceptional leaf spot resistance, thereby reducing the number of fungicide applications needed to produce a successful crop. While this initial work will focus on leaf spot, it is envisioned that a similar approach could be employed to develop lines for other economically important diseases such as Sclerotinia blight, tomato spotted wilt virus (TSWV), Cylindrocladium black rot (CBR), and stem rot (Sclerotium rolfsii)).
New tools and resources to identify mechanisms of pre-harvest aflatoxin resistance in peanut – Genetic variant discovery and marker validation
Principal Investigator: Dr. Alicia Massa, USDA-ARS, Dawson, GA
Aspergillus flavus and A. parasiticus are opportunistic pathogens that invade peanut seeds causing accumulation of aflatoxins. Sorting and removal of aflatoxin-contaminated kernels cost farmers and the peanut industry millions of dollars every year. Therefore, breeding for reduced aflatoxin accumulation is a high priority. Current breeding methods for aflatoxin-resistant are genetically inefficient due to limited understanding of the underlying mechanisms of aflatoxin accumulation. We propose to develop new tools and resources to identify these mechanisms. The specific objectives for this project are to: a) identify genetic variants in the peanut transcriptome in response to Aspergillus infection, and b) validate molecular markers associated with aflatoxin-resistant germplasm. We anticipate that results from this research will have immediate application in genome association analysis, genetic introgression, pre-breeding/breeding, and assessment of resistant candidates at pre-harvest. In addition, the genetic variants detected in this study will provide a valuable tool for target design in genome editing.
We have generated transcriptome data (RNA-seq) and characterized gene expression changes in the peanut-Aspergillus interaction of susceptible vs. resistant genotypes. The specific objectives for this project are to: a) identify genetic variants, primarily single nucleotide polymorphisms (SNPs) and small insertions/deletions (InDels) in the peanut transcriptome in response to Aspergillus infection, and b) validate SNPs/InDels based markers associated with aflatoxin-resistant germplasm.
Characterization of peanut leaf pathogens for varietal development in the USA
Principal Investigator: Dr. Soraya Leal-Bertioli
Plant pathogens and pests place high production constraints in the U.S. peanut production. It is clear that cultivars that are developed to be resistant to a particular pathogen (LLS/ELS) do not hold resistance in every environment or every growth season. It is not known if this id due to environmental changes, host genetic instability or change in population structure. To date, no systematic characterization has been done for these pathogens in order to address these questions. With previous funds, we have collected over 300 samples from five states in the USA. Over 200 isolates were successfully recovered and are now being kept in long term storage. Therefore, we have created an extensive and diverse mycological bank that is held at UGA and is available researcher who requests it. In this proposal, our goal is to characterize diversity within the agents of late and early leaf spot disease. A better understanding of diversity within the pathogen populations will provide valuable insights for further epidemiological studies, disease management strategies and approaches to breeding for resistance to mitigate constraints imposed by these biotic stresses on peanut.
The characterization of USA Arachis wild species accessions and making their genetic diversity available for peanut breeding by the creation of a structured wild tetraploid germplasm core
Principal Investigator: Dr. Soraya Leal-Bertioli
The genus Arachis contains 82 described species. The botanical section Arachis is of particular interest because it includes cultivated peanut, an allotetraploid, and closely related wild species, most of which are diploids. The main collections of wild Arachis species in the USA are at Plant Genetic Resources Conservation Unit (PGRCU), USDA, Texas A&M and NCSU. Together, these collections hold around 75 out of all Arachis species. Diploid species of the Arachis section have traits that can be transferred to cultivated peanut through hybridization methods and can be of particular agronomic interest (eg. resistance to pests and diseases, larger seed, productivity, drought tolerance, etc). Several cultivars derived from wild relatives have been released (Webb, Tifguard, Georgia 14N, etc), with higher disease resistance, thus saving farmers resources.
Arachis taxonomy is difficult and complex. There isn’t complete consensus about placement of accessions within species or about the existence of some species as biological entities or the proper differentiation of ‘twin’ species from seed characteristics only. Occasionally, seed mixture happens and the error can be propagated to many generations down the line, creating confusion and adding extra difficulties to the already arduous job of pre-breeding. The difference in ploidy between wild species and peanut hinder transfer of useful alleles to the crop.
In the first year of this project, we genotyped all known species from the Arachis section plus some selected representatives of other eight sections using 50k SNPs that cover the whole genome. This gave us a clear idea of phylogentic position of each species, correct placement of several accessions, as well as the chance of identifying seed mix and mislabelings. The visit by Dr. Valls did not happen due to COVID 19. The resources were directed to genotyping more accessions, thus completing all the Arachis section and having representatives of all the other sections.
We propose (i) to genotype accessions of Arachis species in the PGRCU-USDA genebank, that represent all the remaining species of the genus Arachis Arachis Axiom chip. With this, we will be building on the database that will help understand the structure of the genus and serve as species ‘barcodes’. (ii) Diploid species of the Arachis section have traits that can be transferred to cultivated peanut through hybridization methods and can be of particular agronomic interest (eg. resistance to pests and diseases, larger seed, productivity, drought tolerance, etc). We propose to create a core collection as strategic reserve of newly induced allotetraploids. These allotetraploids will be made available to the all U.S. peanut breeding programs through the National Plant Germplasm System.
PeanutBase.org, a Peanut Genetic and Genomic Toolbox
Principal Investigator: Dr. Sudhansu Dash
The PeanutBase has successfully evolved over the years into its current state in integrating major data types from genetic and genomic researchers. This has been concomitant with the progress in research and data generated by the peanut research community over these years starting from the Genomics Initiative and with full support from The Peanut Research Foundation. As a result, tools are in place to explore the ancestral diploid and tetraploid genomes along with genetic maps and markers from the genotyping chip (Affy 48k SNP set); side-by-side visualization/comparison of genotype data; similarity between regions on the peanut genome and related genomes; browsing and mining PeanutBase data; interactive geographic exploration of germplasm accessions; and phenotypic data visualization of the peanut mini core collection. Additional tools are being integrated into PeanutBase to view GWAS results across multiple traits and gene expression comparison with other legume species and provision for adding new genome assemblies.
A continued objective of Peanutbase has been integrating various genomic and genetic data types and making them useful for breeding programs. With more and more phenotypic experiments and data in the horizon along with GWAS data in the coming years, PeanutBase will focus on incorporating these data into PeanutBase in a way such that various tools can pick them up and help users explore and use them in a trait centric way. This will mainly involve incorporating phenotypic, GWAS, expression and genomics data as they are generated by the community.
The proposed work will continue to enhance (1) ease of scouting marker association with traits (2) germplasm and their phenotype data exploration for breeding programs (3) access to cultivated and wild peanut genomes.
Marker-Assisted Backcrossing of Breeding Lines for Drought Tolerance, Improved Grade, High-Oleic Oil, and Resistance to Root-knot Nematodes
Principal Investigator: Dr. Mark Burow, Texas AgriLife Research
Our objectives are to:
- Develop a marker-assisted program to produce cultivars with enhanced drought resistance.
- Define causes of yield reduction associated with drought, identify peanut genotypes which use water more efficiently and tend to yield better under drought conditions, and/or develop markers associated with sustainable yields under drought stress.
- Associate molecular and physiological markers with specific mechanisms of water efficiency and drought tolerance.
Genotyping by Resequencing – A Community Resource for Inexpensive Genotyping and for Study of Gene Expression to Identify QTLs in New Peanut Breeding Populations.
Principal Investigator: Dr. Mark Burow, Texas AgriLife Research
This project will develop a set of 2000 highly-polymorphic SNP (Single Nucleotide Polymorphism)-based markers that can be genotyped in a single sequencing reaction for a large (96 to 384) number of individuals in a breeding population.
This will provide an inexpensive method of scoring a minimum number of SNPs needed for identifying markers for QTLs (Quantitative Trait Loci) in new breeders’ populations. We have a quote that will allow for a cost of $13 per sample ($8 per sample for library preparation plus ca. $5 for sequencing) after the initial development cost is paid. This will allow an inexpensive genotyping platform for QTL analysis in new breeding populations, sufficient for low-cost identification and validation of new QTLs at a minimal marker density. The open nature of the assay allows for inexpensive addition of new targets if specific needs arise. The Axion_Array2 SNP chip or genotype-by-sequencing (GBS) can be used thereafter if a higher-resolution marker map is needed.
Our objective is to develop genetic resources that will move the peanut community forward to exploit germplasm for disease, drought, aflatoxin resistance, or improved flavor profiles.
Transcriptome Analysis of Wild Species Peanut under Induced Drought Stress
Principal Investigator: Dr. John Cason, Texas AgriLife Research
This project focuses on the development of germplasm by the identification of drought tolerance genes in wild species peanut. Groundwater depletion, climate change and population expansion are a growing concern in many areas of the world. For example, groundwater levels are reaching critical levels in the West Texas growing region; in response, this project involves research for the introduction of increased drought tolerance using wild species peanut germplasm into the cultivated peanut. We propose to validate a transcriptional analysis of Arachis ipaënsis Krapov. and W.C. Gregory (KGBPScS 30076) (B genome donor), and A. dardani Krapov. and W.C. Greg. (GK 12946) that was conducted in previous research with other funding. In addition, we will expand the study to examine the transcriptome of A. dardani Krapov. and W.C. Greg. (VKVeSvS 7215) for presence of the previously identified candidate genes associated with drought tolerance. A replicated trial will be conducted where drought will be imposed in controlled greenhouse conditions. Subsequently RNA leaf and root tissue will be extracted and used to confirm the presence of the candidate genes using differential gene expression analysis as well as Reverse Transcription qPCR to validate the previous findings.
A critical problem in developing drought tolerance is the identification of genes and the eventual transfer of those genes into elite cultivated material. This project will capitalize on the vast expertise of the Texas A&M AgriLife Research Peanut Breeding program’s drought tolerance and wild species research experience. The tools and germplasm once fully developed will addresses the Peanut Research Foundation (PRF) goals of development of molecular markers for specific mechanisms of water efficiency and drought tolerance as well as to develop molecular markers associated with sustainable yields under drought tolerance. This research will build on previous research that identified 14 candidate genes for drought tolerance to later serve as potential targets for gene introgression and marker development. This project also addresses the PRF goal of developing populations with valuable traits introgressed from wild species. This study is being conducted in an effort to validate findings from previous research that identified the presence of up or down regulated genes. We will use both high throughput sequencing and analysis as well as Reverse Transcription qPCR to study the wild species A. dardani and A. ipaënsis to accomplish the project goals. In addition to the original species and specific accessions used in the original study, we will expand the project to include the analysis of the transcriptome of a related accession of A. dardani Krapov. and W.C. Greg. (VKVeSvS 7215). By including the new accession, we will be able to evaluate how well conserved the genes are in the related accessions. Although not directly included in this plan of work, the result will eventually be used to develop molecular markers that can be used in conjunction with an introgression pathway that is also under development to move the genes into cultivated peanut. The specific objectives are:
- Validate previously identified drought tolerance candidate genes identified in A. ipaënsis (KGBPScS 30076) and A. dardani (GK 12946) by repeating the previous study using RNA-seq and Reverse Trascription qPCR.
- Expand the imposed drought study to include an additional accession involved in the project consisting of A. dardani (VKVeSvS 7215) to determine their conservation in the genome, identify new candidate genes.
Special Aflatoxin Call for Proposals: Genetic Approach to Mitigate Aflatoxin Contamination in Peanut
Peggy Ozias-Akins: Institute of Plant Breeding, Genetics, & Genomics, University of Georgia
Josh Clevenger: HudsonAlpha Institute for Biotechnology, Huntsville, AL
Aflatoxin is a highly carcinogenic mycotoxin produced by the fungi Aspergillus flavus and A. parasiticus upon invasion of seeds and exposure to stress. Peanut is particularly susceptible to preharvest aflatoxin contamination when plants are exposed to late season drought or postharvest contamination when seeds are improperly stored. While aflatoxin contamination is adequately managed in the US by inspection of peanut lots at buying points and after storage and analysis of food ingredients and manufactured foods, each of these interventions is costly and reduces profit for each sector in the value chain. Genetic resistance would contribute greatly to mitigating costs and hazards associated with aflatoxin contamination.
Recent and reproducible results using an in vitro seed colonization protocol have reliably distinguished tolerant from susceptible lines of peanut. A reduction of aflatoxin contamination in seeds is not correlated with the extent of fungal growth. The in vitro assay has been applied to a segregating population as well as additional germplasm lines with some prior evidence of reduced aflatoxin contamination. We seek to extend these results to parallel pre-harvest evaluation of these lines, genome characterization, and development of breeding materials better adapted to the US growing environment and with increased market acceptability. We predict that the germplasm selected has a range of underlying resistance mechanisms, which can be combined during population development and ultimately selected upon using molecular tools. Discovering associations between molecular markers and reduced aflatoxin contamination, a highly quantitative trait subject to genotype by environment effects, will greatly benefit breeding programs delivering improved cultivars for the peanut industry.
- Develop a reliable and repeatable method to evaluate post-harvest aflatoxin resistance.
- Define mechanisms of pre- and post-harvest aflatoxin resistance and associate molecular markers with specific mechanisms.
- Develop peanut germplasm less susceptible to aflatoxin contamination.