Recipient Organization
DELAWARE STATE UNIVERSITY
1200 NORTH DUPONT HIGHWAY
DOVER,DE 19901
Performing Department
Agriculture and Natural Resour
Non Technical Summary
Blueberry (Vaccinium Section Cyanococcus) is an economically important small-fruit crop native to North America. Most of the northern highbush blueberry (NHB) cultivars have V. corymbosum genetic background. Blueberry production was earlier restricted to temperate regions of Canada and northern parts of the United States. However, more than 38 states, including many of the country's southern states, and several countries worldwide are cultivating blueberry as a commercial crop. The commercial relevance has been steadily growing for the past two decades owing to an increased awareness of the benefits of blueberry to human health. Blueberry plants have stringent growth conditions, and slight increases in the temperature can adversely impact growth and yield. A decrease in plant survival and fruit quality has been observed in some blueberry cultivars when average daytime temperatures exceeded 30°C. Thus, high-temperature stress is a pivotal factor in blueberry production and yield, especially with the high temperatures and increased heat waves anticipated by global warming.The adaptability of blueberry plants to high-temperature stress conditions varies among genotypes. Various southern species of blueberry, such as V. darrowii, are reported to adapt to warmer climates. Such species may carry new genes/alleles that allow the plant to survive and grow in relatively warmer conditions. Identifying such genes/alleles and the development of molecular markers associated with high-temperate stress tolerance traits will facilitate the selection and breeding of new heat-tolerant blueberry cultivars. We used V. darrowii × V. corymbosum cross progenies (320 plants) for genome-wide association study (GWAS) with genotyping-by-sequencing (GBS) technology. We identified 1,323 single nucleotide polymorphisms (SNPs) significantly associated with high-temperature stress-tolerance traits.In this grant, we propose to develop Kompetitive allele-specific PCR (KASP) and cleaved amplified polymorphic sequence (CAPS) markers for high-temperature stress tolerance in blueberries. We will design the primers for the most significant SNPs and validate their association with the traits. The developed markers will be tested and validated in F2 progenies from the V. darrowii × V. corymbosum cross and 80 available blueberry accessions. Additionally, this project will train postdoctoral research associates and graduate and undergraduate students in modern plant breeding techniques and help build a small-fruit breeding program and research capacity at Delaware State University (DSU).
Animal Health Component
50%
Research Effort Categories
Basic
10%
Applied
50%
Developmental
40%
Goals / Objectives
The main goal of this research is to develop high-throughput SNP-based KASP markers for high-temperature stress tolerance in blueberry that can facilitate marker assisted selection for generating heat-tolerant blueberry genotypes in breeding programs. This goal will be achieved by using the resources generated from our previous studies. We will select SNPs significantly associated with high-temperature stress tolerance and its associated traits, design KASP and CAPS assays for the selected SNPs and validate the designed assays in blueberry F2 population and cultivars.Specific objectives of this project are as follows: 1. Development of SNP-based KASP markers associated with high-temperature stress tolerance in blueberries. 2. Development of SNP-based CAPS markers for high-temperature stress tolerance in blueberries. 3. Validation of KASP and CAPS markers in F2 progenies and blueberry accessions. 4. Integration of the knowledge and resources from this project in teaching/training students and researchers at DSU and local blueberry farmers.
Project Methods
Development of SNP-based KASP markers associated with high-temperature stress tolerance in blueberries.For developing KASP markers, we will select the GWAS-identified SNPs (based on p-value) that are significantly associated with high-temperature stress-tolerance traits. In our preliminary research, we pinpointed 245 SNPs significantly associated with more than one trait and identified 179 candidate genes through sequence BLAST analysis. In our earlier experiment, some of these genes were differentially expressed between V. corymbosum and V. darrowii under heat stress conditions (Callwood et al., 2021). The genes that were commonly identified in GWAS and the earlier transcriptomic study (Callwood et al. 2021) could be important in regulating high-temperature stress tolerance in blueberry species. Therefore, the KASP markers for the selected SNPs could be useful in genotyping and selection in blueberry crossing programs. Apart from these commonly identified SNPs, other SNPs that are significantly associated with important traits such as QY will be selected. We have the physical locations of these SNPs, which will help us select SNPs with significant genome coverage with a better average marker density per chromosome. Only SNPs offering a great contribution to the phenotype variance with significant P values will be selected. We plan to select a total of 300-400 quality SNPs for developing KASP markers to allow for high-throughput genotyping of blueberry populations.Once the genes are selected, and SNP sites are located, primers will be designed from the corresponding sequences. The 100-bp upstream and downstream sequences of the selected SNPs have been extracted from the genomic sequences. For each SNP, two allele-specific forward primers and one common reverse primer based on the flanking sequences around the variant position (SNP) have been designed by using Primer 3 software. The polymorphic SNPs will be converted to KASP markers to test their ability to differentiate the polymorphism by genotyping the two parents. KASP assays (primers and master mix) will be designed according to the instructions by LGC Biosearch Technologies (LGC Genomics) and run on a high-performance LightCycler 480 System (LC480, Roche Life Science; http://www.lightcycler480.com). Markers showing clear allelic discrimination will be selected for further verification to validate their specificity, stability and universality by testing in the F2 population and blueberry accessions.Development of SNP-based CAPS markers for high-temperature stress tolerance in blueberries.For developing CAPS markers, the presence of a restriction enzyme site upstream or downstream of the selected SNP position is essential. For this reason, about a 1- to 1.5-Mb region containing the selected SNP on the respective scaffold sequence will be downloaded from the Draper genome sequence. The selected segment of the genomic sequence will be scanned for restriction sites, and putative alternative restriction sites will be identified by using the programs SNP2CAPS (Thiel et al. 2004) and dCAPS Finder 2.0 (Neff et al. 1998). These programs facilitate the computational conversion of SNPs into CAPS markers. About 300-400 quality SNPs will be screened for their potential for analysis as CAPS markers, and primers will be designed and chemically synthesized. While designing primers, care will be taken to have the largest possible size difference between the digestion products to obtain better resolution on agarose gels. CAPS assays will involve using the DNA from the parental lines initially to check their amplification patterns. Amplicons with clear, single-band patterns of the expected sizes will be digested with the corresponding restriction enzymes according to their reaction conditions prescribed by the manufacturers. PCR, reaction mixture and digestion conditions will be optimized for each marker as per the requirements. If necessary, purification of the amplified product will be carried out before restriction digestion. After amplification, the treated mixture will be run on 1% agarose gels in the TBE buffer, followed by 6X GellRed (Biotium Inc., USA) staining to check the digestion patterns. The undigested (control) and digested samples from the same sample will always be placed side-by-side on the agarose gel for better comparison. The markers giving clear amplicons and subsequent complete or partial digestion will be selected for further validation among F2 progenies and blueberry cultivars to determine their association with the trait.During the subsequent validation experiments with the F2 populations, we may find some plants heterozygous for a particular SNP. Hence, the ability of the respective CAPS markers to distinguish between heterozygotes and homozygotes will also be checked. For this, equal concentrations of DNA samples from two different homozygous plants will be mixed to simulate the heterozygous one and check for digestion patterns.Validation of KASP and CAPS markers in F2 progenies and blueberry accessions.The designed KASP and CAPS markers will be validated initially in F2 progenies from the BNJ16-4 and BNJ16-5 crosses. Leaf samples from young, actively growing blueberry plants will be collected in dry ice, and DNA extraction will be performed with a commercially available plant DNA extraction kit. DNA samples of good quality (quantified by using the Qubit Fluorometer) will be used for the experiments. Marker validation in these crosses will further confirm their association with the respective trait. However, it is important to validate the SNPs in blueberry germplasm with different genetic backgrounds, strengthening the association of the SNP and widening its applicability. For this, we will use 80 widely used blueberry accessions that are growing in the DSU research and outreach center. Several cuttings from these plants will be propagated in 3.5" × 5" small pots and maintained in the greenhouse for use in validation experiments. In addition, 2-year-old plants of the blueberry cultivars that are well adapted to warmer climates will be obtained from local nurseries. Together, about 100 blueberry genotypes with different genetic backgrounds will be used for further validation of the developed markers. To determine their phenotypic response in high-temperature stress conditions, the plants will be subjected to high-temperature stress in the growth room and phenotypically screened for high-temperature stress-tolerance traits, including electrolyte leakage, reactive oxygen species, chlorophyll fluorescence and malondialdehyde content. Newly matured leaves will be used for measuring photosynthetic and physiological parameters. DSU has the required greenhouse and growth chamber facilities that can be used for phenotyping and validation purposes. Once phenotype responses are measured, they will be screened for marker assays to validate their association with the respective SNPs. These validation experiments will help in examining the application of developed markers across genetic backgrounds.