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Abstract
Cereals high in amylose content (AC) and resistant starch (RS) offer potential health benefits. Previous studies using chemical mutagenesis or RNA interference have demonstrated that starch branching enzyme (SBE) plays a major role in determining the fine structure and physical properties of starch. However, it remains a challenge to control starch branching in commercial lines. Here, we use CRISPR/Cas9 technology to generate targeted mutagenesis in SBEI and SBEIIb in rice. The frequencies of obtained homozygous or bi-allelic mutant lines with indels in SBEI and SBEIIb in T0 generation were from 26.7 to 40%. Mutations in the homozygous T0 lines stably transmitted to the T1 generation and those in the bi-allelic lines segregated in a Mendelian fashion. Transgene-free plants carrying only the frame-shifted mutagenesis were recovered in T1 generation following segregation. Whereas no obvious differences were observed between the sbeI mutants and wild type, sbeII mutants showed higher proportion of long chains presented in debranched amylopectin, significantly increased AC and RS content to as higher as 25.0 and 9.8%, respectively, and thus altered fine structure and nutritional properties of starch. Taken together, our results demonstrated for the first time the feasibility to create high-amylose rice through CRISPR/Cas9-mediated editing of SBEIIb.
Highlights
Diet-related non-infectious chronic diseases, such as coronary heart disease, diabetes, and certain colon and rectum cancers, are major causes of morbidity and mortality in both developed and developing countries (Regina et al, 2006; Chen et al, 2012)
By using a japonica rice variety Kitaake as an example, we here further demonstrated the advantages of CRISPR/Cas9-mediated genome editing in crop improvement and presented a better alternative strategy to breed for high amylose rice to meet increasing demand of people who suffer from diet-related non-infectious chronic diseases
Down-regulation of SBEII in potato leads to an increase in amylose to approximately 35% (Jobling et al, 1999), whereas potato starch with an amylose content (AC) of 70% or higher was yielded by co-suppression of SBEI and SBEII (Schwall et al, 2000)
Summary
Diet-related non-infectious chronic diseases, such as coronary heart disease, diabetes, and certain colon and rectum cancers, are major causes of morbidity and mortality in both developed and developing countries (Regina et al, 2006; Chen et al, 2012). Around 600 million people are estimated to live with diabetes in 2035 (Unwin et al, 2013). A cereal grain higher in amylose content (AC) is always a good source of RS (Jiang et al, 2010). Cereals high in RS are benefit to improve human health and to reduce the risk of those serious non-infectious diseases (Regina et al, 2006).
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