Source: N Y AGRICULTURAL EXPT STATION submitted to NRP
MAPPING THE GENETIC BASIS OF FIRE BLIGHT RESISTANCE IN MALUS SIEVERSII, THE PRIMARY PROGENITOR SPECIES OF APPLES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1030855
Grant No.
2023-67011-40495
Cumulative Award Amt.
$120,000.00
Proposal No.
2022-11314
Multistate No.
(N/A)
Project Start Date
Jun 15, 2023
Project End Date
Jun 14, 2025
Grant Year
2023
Program Code
[A7101]- AFRI Predoctoral Fellowships
Recipient Organization
N Y AGRICULTURAL EXPT STATION
(N/A)
GENEVA,NY 14456
Performing Department
(N/A)
Non Technical Summary
This researchaims to generate knowledge and tools to better breed apples with resistance to the destructive bacterial disease, fire blight. The focus is onthe primary progenitor ofdomesticated apples,M. sieversii.This wild species has moderately sized applesand genotypes with strong fire blight resistance that can be used develop fire blightresistant apple cultivarsin less generations.To achieve this,we aim tofinemapa previously identified moderate effect fire blight resistance allele, Msv_FB7. A large genetic mapping population, artificial fire blight inoculations and adense panel of genetic markers will enableamore precisecharacterizationthe location of Msv_FB7. Low-cost markers will then be developed with that knowledge to allow apple breeders to utilize that source of genetic resistance in breeding programs. Additionally, three genetic mapping populations made from crossinga highly susceptible apple cultivarwith several fire blight resistantM. sieversiigenotypes will be used to identifygenetic regions withfire blight resistance alleles. The offspringof these crosses will be screened with a previously developed fire blight leaf disc assayto rapidly find the most resistant and susceptible individuals at the seedling stage.Then, several of the most resistantand suseptible individuals will be separately pooledforDNA and RNA sequencing to identify the genetic basis of fire blight resistance in these plants. The results of these projectscanbe used to develop new apple cultivars withfire blight resistance that are less resource intesive to manage.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20111101080100%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1110 - Apple;

Field Of Science
1080 - Genetics;
Goals / Objectives
The main goals of the research are focused on understanding the genetic basis of fire blight resistant from M. sieversii. Objective 1 aims to fine map a major effect fire blight resistance QTL, Msv_FB7, from M. sieversii, which will improve knowledge for apple breeders to develop fire blight resistant apple cultivars. Objective 2 is to develop low-cost markers that are tightly linked to Msv_FB7, providing an accessible tool for apple breeders to develop fire blight resistant apple cultivars. Finally, Objective 3 seeks to identify novel QTL from additional highly resistant M. sieversii accessions. This objective will involve the development of three new populations for genetic mapping of fire blight resistance, screening the population using a previously developed image-based leaf disc assay. Bulk groups will be generated fromsusceptible and resistant genotypes for a multi-omic bulk segregant analysis andmap novel sources of fire blight resistance to be used for future apple breeding efforts.
Project Methods
Objective 1: Fine map a major effect fire blight resistance QTL from M. sieversii, Msv_FB71.1 Genetic Mapping Populations: To fine map the major effect QTL on LG07 of M. sieversii, I performed controlled crosses in May 2022 with three resistant F1 progeny from GMAL4591 (PI613959) as paternal parents crossed to the fire blight susceptible cultivar, M. domestica cv. 'Idared' (PI588841). I have already extracted seeds from fruit obtained from these crosses to generate a mapping population of 1,200 F1 individuals.1.2 Recombinant Identification and Sequencing: Recombinants for Msv_FB7 from the full population will be identified through genotyping of the parents and progeny with 5-7 SSR markers developed by our lab through fragment analysis. Primers for the AmpSeq markers will be designed from conserved sequences in the QTL region with the Gala and M. sieversii phased diploid reference genomes. Recombinant and parental DNA will be sent for amplicon sequencing.1.3 Fine Mapping Analysis: AmpSeq reads from recombinant individuals and parents will be filtered and aligned to the M. sieversii phased diploid reference genome. M. sieversii parental alleles and recombination events will be more precisely tracked across the region with the high-density panel of AmpSeq markers. The goal will be to reduce the 8cM region of interest to at least 3cM for further marker development. A subset of recombinants will be replicated via bud grafting and used for fire blight inoculations to confirm the resistance levels of the genotypes.Objective 2: Develop low-cost markers tightly linked to Msv_FB72.1 KASP Marker Design: Fine mapping from Objective 1 will identify key AmpSeq markers in tight linkage with the Msv_FB7 QTL, these will be targeted for KASP marker design. Primer pairs from the AmpSeq reads will be used to design primer pairs where the terminal base of the forward primer is the polymorphism of interest. The primer sequences for each proposed KASP marker will be aligned to the Golden Delicious Double Haploid genome as well as the phased diploid Gala and M. sieversii genomes. Primers with the fewest indels in their sequences and the highest potential transferability will be selected for further marker testing. The KASP markers will be validated using susceptible and resistant recombinants and parents from Objective 1 and a subset of genotypes from the original QTL population, GMAL4591, to evaluate their fidelity.Objective 3: Identify novel QTL from additional highly resistant M. sieversii accessions3.1 Plant Populations: I performed crosses in May 2022 to generate new biparental F1 populations with three highly resistant M. sieversii accessions (PI657054, PI657085, PI657115) crossed with highly susceptible M. domestica cultivars ('Gala', 'Golden Delicious'). Approximately 250-300 seeds from each population will be planted in the greenhouse.3.2 Phenotyping and Bulk Generation: Fire blight screening of seedlings will be performed using the previously described detached leaf disc assay method, I developed (Figure 4). A single BLUP value for each genotype will be used for ranking the most susceptible and resistant genotypes. A subset of progeny with extreme phenotypes will be bud grafted to a standard rootstock with 4 replicates and planted in the greenhouse. Young shoots of the bulked populations will be inoculated with E. amylovora using the standard dipped scissor method, percentage of shoot lesion length (PSLL) will be collected at three time points. The area under the disease progress curve (AUDPC) will be used to validate resistance classifications.3.3 DNA-RNA Sequencing: Young leaf samples from two-time points of infection (control- 0hpi; symptomatic-48hpi) will be collected. DNA from each parent and progeny bulks will be sent for library prep, pooling, and whole genome sequencing (WGS). Total RNA from parents and progeny bulks will be sent to be library prepped, pooling, and sequenced using 3'RNA-seq.3.4 Genetic Mapping Analysis: The genotype data collected will be trimmed, cleaned, and SNPs will be called using the M. sieversii phased diploid reference genome. BSA-seq (bulk segregant analysis) and BSR-seq (bulk segregant with RNA) will be performed with genomic markers and differentially expressed genes (DEGs). Integrated genomic and transcriptomic analysis will follow Kremling et al. 2019, where SNPs are assigned to the nearest gene, filtered for genes used in the transcriptomic bulk segregant analysis, and combined genome/transcriptome p-values will be calculated with the Fisher's Combined Test (FCT).

Progress 06/15/23 to 06/14/24

Outputs
Target Audience:The target audience of my projects include: Apple genetics/genomics research community Applied apple breeders and breeding programs The greater community of researchers working on disease resistance in long-cycle woody perennials This funding period I was able to reach and disseminate my research to: Researchers working on genetics and genomics of tree fruit crops at scientific crop genetics conferences who are interested in the methods and outputs of this research. Several apple breeders from the major US apple breeding programs including James Luby at the University of Minnesota and Kate Evans from Washington State University. These breeders were interested in incorporating my markers into their breeding program to try and utilize the fire blight resistance QTL Msv_FB7 in their programs. Commercial apple growers in New York State interested in how the downstream output of this research will help to develop fire blight resistant apples. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The funding from this USDA-NIFA predoctoral fellowship has enabled the PD to attend the National Association of Plant Breeders conference in both 2023 and 2024. This conference is a critical step in connecting the PD to a larger network of researchers in the field to make connections and improve the prospects of a search for post-PhD positions. Additionally, this funding source enabled the PD to attend the week-long short course entitled "Multi-omic Integration for AI Genomic Prediction Breeding Under Different Approaches: Past, Present and Future" hosted by the University of Florida in 2023. This short course was both a high-level intensive on quantitative genetics and served as an excellent networking outlet to engage with a group of international researchers teachings and attending. How have the results been disseminated to communities of interest?The outputs of this research fellowship have been presented at conferences and symposiums as both oral and poster presentations. Firstly, the current results of Objective 1 have been presented as an oral presentation in June 2024 at the Cornell SAGES Symposium at Cornell AgriTech in Geneva, NY. In July 2024, this work was also presented as a poster at the National Association of Plant Breeders (NAPB) conference in St. Louis, MO. At the NAPB conference, the PD was able to engage directly with stakeholders of this work, meeting and discussing the findings with the breeders leading the major public apple breeding programs in Washington and Minnesota. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Fine map a major effect fire blight resistance QTL from M. sieversii, Msv_FB7 This objective is largely completed though further genetic analysis to narrow the region of interest will be conducted. Additionally, a comprehensive bioinformatic analysis of candidate genes is underway to support these fine mapping efforts. Objective 2: Develop low-cost markers tightly linked to Msv_FB7 Using the SNP panel for the DarTag platform we have developed several SNPs have been targeted for conversion to KASP markers. We plan to order those markers and back screen all of the parents, grandparents and progeny of the fine mapping population to test the efficacy of the markers. The paper that encompasses objective 1 and objective 2 is currently drafted is planned to be published by the end of the Year 2 project term. Objective 3: Identify novel QTL from additional highly resistant M. sieversii accessions A second round of Erwinia amylovora inoculations will be performed on these bulk segregant populations to improve the statistical power of the downstream analysis. RNA-sequencing will be finished at the beginning of the Year 2 term as well as the bioinformatic analysis to identify differentially expressed genes across the mock inoculated and inoculated groups tested. The fully integrated bulk segregant analysis that incorporates the phenotypic data, genome-wide SNP markers, and RNA-seq data will be performed in Year 2. By the end of the Year 2 term we plan to have the manuscript for this objective written and finalized for publishing in a peer-reviewed journal.

Impacts
What was accomplished under these goals? Objective 1: Fine map a major effect fire blight resistance QTL from M. sieversii, Msv_FB7 The pseudo backcross population used for fine mapping was screened with the previously developed flanking markers for Msv_FB7. From 832 progeny 35 recombinants were identified to be used for further genetic analysis. The recombinants as well as the relevant parents and grandparents of this population have been sent for genotyping with a newly developed DarTag SNP genotyping platform with the Dart company. This provided 3K SNPs genome wide and 100+ SNPs across chromosome seven that are being used for a fine mapping analysis to narrow the region of interest further. The recombinant individuals as well as the parents and grandparents have been clonally replicated to 6-7 replicates grafted on the same rootstock genotype. These plants have been inoculated once with Erwinia amylovora and measured over three time points to calculate the area under the disease progress curve as a metric of disease severity. Inoculations with these plants are being repeated currently to strengthen the statistical power of the fine mapping analysis. Objective 2: Develop low-cost markers tightly linked to Msv_FB7 A newly developed panel of pan-generic SNPs across different Malus species were selected for DarTag genotyping. The SNPs in the region of interest of Msv_FB7 have been selected for conversion to KASP markers. The materials for the development of these KASP markers have been ordered and the remaining goals for this objective will be completed in Year 2. Objective 3: Identify novel QTL from additional highly resistant M. sieversii accessions Over 600 seedlings from three F1 biparental populations were screened using the image-based leaf disc assay to identify 30-40 progeny from each population that are in extreme groups of susceptibility or resistance to Erwinia amylovora. These seedlings and the parental genotypes were grown out and used to replicate each progeny into 8-10 clonal replicates, all on M7 rootstocks. These grafted populations were inoculated one time with Erwinia amylovora and has three time points of data taken during the infection period. All of these genotypes were sent to be genotyped using the DarTag genotyping platform to give ~3K genome-wide SNPs for bulk segregant analysis. Additionally, during the first round of inoculation, leaf samples were collected and frozen for RNA extraction. RNA extractions are now completed for all samples and soon to be sent for Illumina RNA-sequencing. Altogether, this data will contribute to the multi-omic bulk segregant analysis planned to be completed in Year 2.

Publications