Location: Sunflower and Plant Biology Research
Project Number: 3060-21220-033-007-T
Project Type: Trust Fund Cooperative Agreement
Start Date: May 1, 2020
End Date: Dec 31, 2023
Objective:
This project is a continuation from the previous proposal where we identified a heterotic locus in the F1 hybrids between ‘Westar’ (canola, Brassica napus L.) and its chromosome segment substitution line (CSSL) introgressed with a chromosome segment in A10 of ‘Surpass 400’. The F1 hybrids, ‘Westar’ × CSSL and CSSL × ‘Westar’, increased 7% and 20% in grain yield compared to the better-parent value respectively under the growth chamber conditions. To map the heterotic gene further, we developed a set of new introgression lines (ILs: IL 003, IL 005, IL 008, IL 009, IL 0013, IL 017, IL 035, IL 052) carrying different lengths of introgressed chromosome segments in A10 of ‘Surpass 400’. Eight ILs were crossed with Westar reciprocally. The resultant 16 F1 hybrids along with their parents (ILs and Westar) were planted for three years (2017: Fargo and Prosper, ND; 2018: Fargo and Langdon, ND; 2019: Fargo) to estimate heterotic effects. Based on phenotypic data (grain yield of 16 hybrids compared to the better-parent value) and genotype data (regions of those introgressed segments and genotyping-by-sequencing data), we have confirmed that the heterotic locus is within the 26-Kb region in chromosome A10.
The overall goal of our unit research is to develop effective, environmentally sound weed management strategies in northern regions of the U.S. using winter oilseed crops that provide additional cash value to the farmer and provide ecosystem benefits similar to cover crops. Development of higher yielding winter- and/or spring-canola would greatly impact U.S. canola acreage and yield, while also providing value-added ecosystem benefits including weed suppression and pollinator habitat. Our current task is to determine the functions of candidate heterotic gene(s) within the mapped 26-Kb region and shed light on the molecular mechanisms regulating heterosis. To accomplish this goal, our objectives are: 1) sequence the 26-Kb region to confirm the predicted candidate genes, and 2) evaluate the roles or these candidate genes in canola based on gain-of-function approach by over-expressing the candidate genes and based on gene knockout approach using CRISPR/Cas9 technology.
Approach:
Objective 1. Sequence the 26-Kb region: The heterotic locus has been mapped within the 26-Kb region based on three years’ grain yield results and GBS analysis. To affirm the sequence of this 26-Kb region, several pairs of PCR primers have been designed for the amplification of a series of overlapping DNA fragments in Westar with size ranging from 4- to 7-Kb in length. These primers will be used to amplify DNA fragments in Westar, CSSL, IL 009, and IL 013. The amplified DNA fragments from these 4 genotypes will be gel purified for Illumina and/or Nanopore DNA sequencing to determine the cross over sites and the introgressed sequence within the 26-Kb region. Primer walking method will also be used if some introgressed DNA segments cannot be amplified with abovementioned PCR primers in CSSL, IL 009, and IL 013.
Objective 2. Functionally test the roles of these candidate genes: Two approaches will be applied for functional analyses: 1. over-expression of candidate genes to determine gain-of-function phenotype, and 2. gene knockout to determine loss-of function phenotype. For gain-of function approach, we will alter phenotypic changes through expression of transgenes under the control of a strong promoter. Candidate genes will be PCR amplified from the CSSL line using specifically designed primer pairs. The amplified PCR products will be cloned into a standard cloning vector individually following the manufacturer’s instructions. These sequences will then be cloned into the plant expression binary vector pCAMBIA S1300 independently under the control of a super promoter, and the constructs will be transferred into Westar genome through Agrobacterium-mediated transformation. pCAMBIA S1300 also contains a hygromycin gene as a plant selectable marker under the control of a cauliflower mosaic virus (CaMV) 35S promoter. After transforming the constructs into the Westar genome, the function of each candidate gene can be observed based on the development of added heterotic effects, without the need to be a hybrid.
For gene knockout approach, we will use CRISPR technology to target the unique sequences present in candidate genes and generate canola knockout mutants. We will design paired guide RNAs based on the sequence of candidate genes. The use of CRISPR paired guide RNAs will increase on-target cutting efficiency and can produce genomic deletions. Paired guide RNAs will be cloned into the intermediate vector pCBC and further cloned into the pCAMBIA-derived binary vectors with a hygromycin-resistance gene as a selectable marker using the protocol for dicot plants. Recombinant plasmids confirmed by sequencing will be used to transform the CSSL line. CRISPR-Cas9-mediated genome editing (indels) will be confirmed by PCR and subsequent sequencing of the PCR products from the T1 lines. Transgenic lines with confirmed deletions will then be used to test for heterotic effect based on phenotypes of the cross between the genetically modified CSSL and Westar.
