| Autor: |
Andrew Farmer, Juan J. Gutierrez-Gonzalez, Shaun J. Curtin, Melinda R. Dornbusch, Susan S. Miller |
| Rok vydání: |
2021 |
| Předmět: |
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| Zdroj: |
The Alfalfa Genome ISBN: 9783030744656 |
| DOI: |
10.1007/978-3-030-74466-3_16 |
| Popis: |
Medicago sativa (alfalfa) is a perennial outcrossing tetraploid flowering legume plant and the third most widely grown crop in the US, approximately 80 million acres. Alfalfa is an important cover and forage crop used for grazing, hay, and silage in many parts of the world and is the main forage crop for meat and milk production. Not surprising, there is interest in the use of new breeding technologies (NBT) including gene editing to improve various traits such as yield and stress responses. The CRISPR/Cas9 reagent is a highly effective gene editing tool that can generate site-directed double-stranded breaks (DSB) that results in frame-shift mutations. In this chapter, we describe the construction of a CRISPR/Cas9 reagent that targets the alfalfa Pho2 genes. Plants recovered from tissue culture were screened for targeted mutations using either cleaved amplified polymorphic sequences (CAPS) or the target amplicon clone and sequencing assays (TACAS). We found these techniques to be sufficient for screening targets in diploid plants but less effective at screening multiple tetraploid targets. Therefore, PacBio® long amplicon sequencing was evaluated for mutant characterization in alfalfa. The sequence data was processed using the SMRT link platform and analyzed using a workflow on the Geneious® software. Using these tools, hundreds of plants could be rapidly genotyped with approximately 57% of plants exhibiting targeted mutations. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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