Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061
Performing Department
(N/A)
Non Technical Summary
While soybean is the richest and least expensive source of plant-based protein, it has several seed compounds that affect animal nutrient digestion and absorption. The main antinutritional factors are trypsin inhibitors (TI) and lectins (LEC). The team has made great accomplishments on these antinutritional factors such as phenotyping, gene discovery, mutant development and gene editing. There is a critical need to continue the research effort to develop improved, commercially viable soybean cultivars with a combination of low TI and LEC. Leaving this need unmet will likely result in an inability to improve animal feed efficiency and decrease energy use in meal processing.The long-term goal of this proposal is to develop and release soybean varieties and germplasms with reduced or removed antinutritional factors with the focus on TI and LEC. Our overall objective in this application is to develop soybean genotypes that have both low KTI and LEC by integrating TILLING by sequencing, gene editing by CRISPR/Cas9, marker-assisted selection, and classical breeding methodologies. Our expected outcomes are new genetic resources with reduced or removed antinutritional factors that will be later offered to private and public breeding programs as parental lines to develop soybean cultivars to improve livestock nutrition in the U.S. These outcomes are thus expected to have a positive impact by providing soybean meal processors and soybean growers with innovative commercial cultivars containing low antinutritional factors that will help improve the feed efficiency of soybean meal for enhanced livestock meat production in the US.
Animal Health Component
30%
Research Effort Categories
Basic
30%
Applied
30%
Developmental
40%
Goals / Objectives
The long-term goal of this proposal is to develop and release soybean varieties and germplasmwith reduced or removed antinutritional factors, while the focus will be on decreasing/removing two of the most undesirable antinutritional factors in soybean seeds: trypsin inhibitors (TI) and lectins (LEC).Supporting objectivesFunctional characterization of the trypsin inhibitors and lectins antinutritional candidate gene using TILLING-by-sequencing+.Gene editing of the highly seed-expressed genes to stack the best two beneficial Kunitz TI (KTI) and LEC alleles.Development of genotypes derived from the mutants with reduced KTI and LEC content through cross and backcross.
Project Methods
Objective 1. Functional characterization of the trypsin inhibitors and lectins antinutritional candidate genes using TILLING-by-sequencing+.Task 1. Library preparation, probe design, and TILLING-by-Sequencing+This step will be carried out in collaboration with Rapid Genomics LLC. (Gainesville, Florida) as described in Lakhssassi et al., 2020. Probes targeting the selected AN candidate gene will be synthesized at high coverage for TbyS+.Task 2. SNP calling, haplotyping and bioinformatics pipeline Briefly, the FASTQ raw reads (text-based format for storing a biological sequence and its corresponding quality scores) will be processed for quality control using FastQC v0.11.9 (Li et al., 2009a,b), while trimming and filtering of low-quality reads will be performed using Trimmomatic V0.3. The clean FASTQ reads will be mapped to the WI82 reference genome using BWA-0.7.17 (Li et al., 2009). Software asset management (SAM) tools v1.10 (Garrison et al., 2012) will be used to filter and sort the bam files to serve as an input for variant calling using Computer Retrieval of Information on Scientific Projects (CRISP) v1.18.0 (Robinson et al., 2011; Ruden et al., 2012). The Variant Call Format (VCF) files generated by CRISP will be filtered by SnpSift and visualized through the Integrative Genomics Viewer (IGV) for demultiplexing. The effect of mutations in each gene will be predicted using the Variant Effect Predictor (VEP) program at Ensembl Plants release 45 (McLaren et al. 2016).Task 3. Develop markers for mutated genesEach soybean mutant identified through Tilling by sequencing will be confirmed first via Sanger sequencing, the corresponding mutation will be used to predict the resulting amino acid change and to develop Kompetitive Allele Specific PCR (KASP) assays as described previously (Shi et al., 2015).Task 4. Analysis of seed KTI and LEC content of the isolated Tilling-by-sequencing+ mutants KTI content will be quantified by HPLC method previously developed by Rosso et al., 2018. We will use sodium acetate (pH 4.5) as an extractant. The KTI in solution will be separated on an Agilent 1260 Infinity series (Agilent Technologies, Santa Clara, CA), equipped with a guard column (4.6 x 5 mm) packed with POROS® R2 10 µm Self Pack® Media and Poros R2/H perfusion analytical column (2.1 x 100 mm, 10 µm). Detection wavelength will be 220 nm.LEC content will be quantified by an HPLC method modified from Anta et al., 2010. We will use sodium acetate (pH 4.5) as an extractant. The LEC in solution will be separated on an Agilent 1260 Infinity series (Agilent Technologies, Santa Clara, CA), equipped equipped with a guard column (4.6 x 5 mm) packed with POROS® R2 10 µm Self Pack® Media and Poros R2/H perfusion analytical column (2.1 x 100 mm, 10 µm). Chromatographic conditions: gradient, 19% B in 0.5min; 19-20% B in 3min; 20-37% B in 0.5min; 37-43% B in 5min; 43-95% B in 0.5min; mobile phases, 0.1% (v/v) TFA in water (mobile phase A) and 0.1% (v/v) TFA in AcN (mobile phase B); flow-rate, 1.5mL/min. Detection wavelength will be 220 nm.Objective 2. Gene editing of the highly seed-expressed genes to stack the best beneficial KTI and LEC alleles.Task 1. Deletion of a major LEC gene in the background of 5-26Gene expression analysis suggests that Glyma.2g012600, named as Lec1e, is highly expressed in soybean seeds but no other plant tissue types. Previous reports also suggest that mutation of Lec1 alone could significantly reduce the LEC content in soy proteins (Goldberg et al., 1993, Orf et al., 1978). Therefore, in this project, we will develop a CRISPR/Cas9 construct carrying guide RNAs targeting Glyma.2g012600. The construct will be transformed into soybean line 5-26 following our well-established gene editing protocol (Wang et al., 2023a). Non-transgenic soybean lines that carry homozygous kti1/3 and Glyma.2g012600 will be identified. SNP markers targeting Glyma.2g012600 will be developed (Wang et al., 2023a). The new soybean line (kti1/3/lec1) will be evaluated for growth performance in greenhouse conditions. The derived soy proteins will be tested for KTI and LEC content.Task 2. Deletion of other KTI genes that may significantly contribute to the KTI content in soy proteinsOur preliminary data also identified three more KTI genes (Glyma.8g341000, Glyma.8g342300, Glyma.8g342200) that are also highly expressed in soybean seeds. Therefore, in this project, we aim to knock out three new KTI genes in the 5-26 background.A CRISPR/Cas9 construct that carries multiple gRNAs targeting Glyma.8g341000, Glyma.8g342300, Glyma.8g342200 will be developed. The construct will be transformed into 5-26. The derived transgenic lines will be genotyped to identify soybean mutants carrying homozygous mutations on all five KTI genes but not the CRISPR/Cas9 transgene. We expected to develop SNP markers associated with each mutant kti gene (Wang et al., 2023a). Marker-assisted selection will be employed to help us to breed the mutant alleles (all five genes or different combinations) into wild-type WM82 and other varieties (Obj. 3). Therefore, we expect to generate a set of near-isogenic lines that carry different combinations of kti mutant alleles. The derived mutants along with wild-type WM82 will be grown in greenhouse conditions, which can be systematically evaluated for the soy KTI content, and future protein digestibility and major agronomic traits. The mutant that carries the best combinations of kti alleles will be used to cross with other elite soybean cultivars including the new mutant line (kti1/3/lec1) as outlined in Task 1.Objective 3. Development of genotypes derived from the mutants with reduced KTI and LEC content through cross and backcross.Task 1 Develop homozygous plants with kti and lec genes using donor parents by TILLING-by-sequencing from Obj. 1Ten parents including five low KTI and five low LEC mutants from Tilling-by-sequencing will be selected to make 25 crosses (KTI × LEC) to develop both low KTI and LEC offspring. Crosses will be made in the greenhouse. Crossing methods, management and harvest will follow our lab crossing protocols. After hybrid seeds are harvested, they will be planted as F1 generation in Blacksburg, VA. True F1 seeds will be harvested and planted in winter nursery as F2 population. DNA will be extracted from each individual plant, and SNPs for mutant genes will be used to screen DNA of all plants. Only homozygous plants with kti and lec genes will be kept, and seeds will be increased for future breeding activities and livestock feeding trials beyond the 3-year project.Task 2 Develop pre-breeding materials using donor parents by gene editing from Obj. 2Ten donor parents with the lowest KTI and LEC content by gene editing from Obj. 2 will be selected to cross with 20 elite breeding lines as recurrent parents that are from Virginia, North Carolina, and Missouri public breeding programs. These varieties are high yielding, resistant to soybean cyst nematode (SCN) or with value-added traits such as high protein content or high oleic acid.Forty cross combinations between 10 donor parents and the 20 elite lines will be designed, and the crosses will be made as described previously. After hybrid seeds are harvested, they will be planted in the greenhouse and then backcrossed with recurrent parents. Harvested BC1 seeds will be immediately planted. DNA will be extracted from each individual plant, and SNPs for mutant genes will be used to screen the DNA of all plants. Only plants with mutant genes will be kept, and other plants will be discarded. Those kept plants will be crossed with recurrent parents for the next cycle of backcrossing and livestock feeding trials beyond the 3-year project.