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CRISPR/Cas9-Based Mutagenesis of Starch Biosynthetic Genes in Sweet Potato (Ipomoea Batatas) for the Improvement of Starch Quality.

ABSTRACT: CRISPR/Cas9-mediated genome editing is a powerful technology that has been used for the genetic modification of a number of crop species. In order to evaluate the efficacy of CRISPR/Cas9 technology in the root crop, sweet potato (Ipomoea batatas), two starch biosynthetic pathway genes, IbGBSSI (encoding granule-bound starch synthase I), and IbSBEII (encoding starch branching enzyme II), were targeted in the starch-type cultivar Xushu22 and carotenoid-rich cultivar Taizhong6. I. batatas was transformed using a binary vector, in which the Cas9 gene is driven by the Arabidopsis AtUBQ promoter and the guide RNA is controlled by the Arabidopsis AtU6 promoter. A total of 72 Xushu22 and 35 Taizhong6 transgenic lines were generated and analyzed for mutations. The mutation efficiency was 62-92% with multi-allelic mutations in both cultivars. Most of the mutations were nucleotide substitutions that lead to amino acid changes and, less frequently, stop codons. In addition, short nucleotide insertions or deletions were also found in both IbGBSSI and IbSBEII. Furthermore, a 2658 bp deletion was found in one IbSBEII transgenic line. The total starch contents were not significantly changed in IbGBSSI- and IbSBEII-knockout transgenic lines compared to the wild-type control. However, in the allopolyploid sweet potato, the IbGBSSI-knockout reduced, while the IbSBEII-knockout increased, the amylose percentage. Our results demonstrate that CRISPR/Cas9 technology is an effective tool for the improvement of starch qualities in sweet potato and breeding of polyploid root crops.

SUBMITTER: Wang H

PROVIDER: S-EPMC6801948 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background and aimsThe tuberization mechanism of sweet potato (Ipomoea batatas) has long been studied using various approaches. Morphological data have revealed that the tuberizing events result from the activation of the cambium, followed by cell proliferation. However, uncertainties still remain regarding the regulators participating in this signal-transduction pathway. An attempt was made to characterize the role of one MADS-box transcription factor, which was preferentially expressed in sweet potato roots at the early tuberization stage.MethodsA differential expression level of IbMADS1 (Ipomoea batatas MADS-box 1) was detected temporally and spatially in sweet potato tissues. IbMADS1 responses to tuberization-related hormones were assessed. In order to identify the evolutionary significance, the expression pattern of IbMADS1 was surveyed in two tuber-deficient Ipomoea relatives, I. leucantha and I. trifida, and compared with sweet potato. In functional analyses, potato (Solanum tuberosum) was employed as a heterologous model. The resulting tuber morphogenesis was examined anatomically in order to address the physiological function of IbMADS1, which should act similarly in sweet potato.Key resultsIbMADS1 was preferentially expressed as tuberous root development proceeded. Its expression was inducible by tuberization-related hormones, such as jasmonic acid and cytokinins. In situ hybridization data showed that IbMADS1 transcripts were specifically distributed around immature meristematic cells within the stele and lateral root primordia. Inter-species examination indicated that IbMADS1 expression was relatively active in sweet potato roots, but undetectable in tuber-deficient Ipomoea species. IbMADS1-transformed potatoes exhibited tuber morphogenesis in the fibrous roots. The partial swellings along fibrous roots were mainly due to anomalous proliferation and differentiation in the xylem.ConclusionsBased on this study, it is proposed that IbMADS1 is an important integrator at the initiation of tuberization. As a result, the initiation and development of tuberous roots seems to be well regulated by a network involving a MADS-box gene in which such hormones as jasmonic acid and cytokinins may act as trigger factors.

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CRISPR/Cas9-Based Mutagenesis of Starch Biosynthetic Genes in Sweet Potato (Ipomoea Batatas) for the Improvement of Starch Quality.

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