Attachment 1 Statement of Work SOW 1232SA25Q0463.pdf

USDA ARS Soybean Genomics and Improvement Laboratory Beltsville Agricultural Research Center

Building 006, Rm 100 Beltsville, MD 20705 Statement of Work (SOW)

Gene Expression and Gene Editing

Study Summary Scientists at the USDA-ARS Soybean Genomics and Improvement Laboratory (SGIL) are working to improve nutrition, seed quality, and other traits in legume crops. Researchers have identified candidate gene variants associated with certain chemical and nutritional traits between varieties and hope to observe the relationship between gene expression and phenotype and changes in phenotype after gene editing to confirm or deny previous research results.

1. Anticipated Period of Performance 10 months-12 months.

2. The work The work involves two separate parts:

a. Use a single vector that can accommodate all four genes to express four genes in rice varieties (Nipponbare or other rice varieties) and corn varieties (KN5585 or other varieties) genotypes, the gene sequence lengths are 5464bp, 1187bp, 2221bp, and 2368bp, respectively. This work will include gene synthesis, vector construction, plant transformation and gene expression validation. We need to obtain T1 generation seeds from at least 10 confirmed positive lines. Confirmation of the success of the four gene transformations is required.

b. Change one base in soybean gene with a sequence length of 1187bp from "A" to "C" (using soybean Williams 82 variety), and obtain T1 generation seeds from at least three mutant lines. Confirmation of the base change is required.

Step 1. Vector Construction

• Gene synthesis o Gene11_2196102_2197289, 1187 bp o Gene7_37166660-37169027, 2368 bp o Gene11_4779592-4781813, 2221 b o Gene2_45475466_45480930, 5464 bp

• Vector construction

• Vector transforming into Agrobacterium strains

Step 2-1. Oryza sativa Transformation [per construct]

• Agrobacterium transformation of Oryza sativa plants [Nipponbare ]

• Positive transformants screening

• Positive transformants screening by antibiotic resistance

• Positive transformants validation by PCR

Milestone

• T1 seeds from at least 10 independent events per construct

• Detailed report including results and methods

Step 2-2. Zea mays Transformation [per construct]

• Agrobacterium transformation of Zea mays plants [KN5585]

• Positive transformants screening

• Positive transformants screening by antibiotic resistance

• Positive transformants validation by PCR

Milestone

• T1 seeds from at least 10 independent events per construct

• Detailed report including results and methods

Sequence Design

• Target gene o Soybean gene locus: Gene11_2196102_2197289

• Targeted single base pair change o Denine (A) to Cytosine (C)

Transgenic Glycine max Line Generation

• Vector Construction o gRNA sequence design o Vector construction and verification

• Agrobacterium transformation of Glycine max plants [Willams 82]

• Positive transformants screening o Positive transformants screening by antibiotic resistance o Positive transformants validation by PCR o Gene editing verification by sequencing

Milestone

• T1 seeds from 2-3 confirmed mutant lines

• Detailed report including results and methods

3. Data and material delivery

a. The gene expression and gene editing protocols and experimental designs will be provided to SGIL scientists.

b. The gene expression and gene editing confirmation test results and related information will be provided to

SGIL scientists.

c. SGIL scientists will provide the gene sequences to the contractor electronically.

d. The contractor will send the T1 generation seeds to the SGIL scientists.