Field assessment of ultra-low asparagine, low acrylamide, gene edited wheat

流行食品中丙烯酰胺的存在已成为食品工业及其供应链面临的最困难的问题之一。丙烯酰胺是一种污染物，由一种称为天冬酰胺的游离（非蛋白质）氨基酸和葡萄糖、果糖和麦芽糖等糖形成。该反应发生在油炸、烘焙、烘烤、烘烤和高温加工过程中。丙烯酰胺被归类为可能导致人类癌症的物质，在高剂量下会影响发育和生育能力。由小麦和其他谷物制成的产品是膳食丙烯酰胺的主要来源。食品中的丙烯酰胺受法规（EU）2017/2158（2018）的监管，该法规设定了食品中丙烯酰胺的基准水平，描述了强制性缓解措施，并要求所有食品企业监测其产品中丙烯酰胺的水平。为限制加工过程中丙烯酰胺形成而开发的方法并不适用于所有产品，可能对产品质量产生不利影响，并且实施起来昂贵。我们的方法是降低农作物产品中丙烯酰胺形成的可能性：对于小麦和其他谷物，这意味着降低谷物中游离天冬酰胺的浓度。该项目源于之前BBSRC资助的工作，在该工作中，我们已经证明了小麦籽粒中游离天冬酰胺浓度的巨大品种差异，并表明它对缺硫和其他营养和环境胁迫因素以及疾病具有高度反应性。天冬酰胺是由一种叫做天冬酰胺合成酶的酶产生的，小麦有五个编码这种酶的基因。其中一个基因TaASN 2已经成为遗传靶点，因为它在谷物中特别活跃。在我们目前的工作中，涉及布里斯托大学Rothamsted Research的团队以及合作伙伴AHDB，KWS，Limagrain，RAGT，Saaten Union和Syngenta，我们使用基因组编辑技术CRISPR-Cas9敲除TaASN 2基因。此外，我们筛选了一个小麦群体，其中突变已被引入化学诱变（一种更古老的技术，自20世纪50年代以来一直用于植物育种），并确定了小麦基因组中TaASN 2的六个版本中的每一个都有突变的品系。这些品系中的TaASN 2突变正在由我们的一个合作伙伴“堆叠”，部分敲除将在项目的第2年完成。其中一个CRISPR系中游离天冬酰胺的浓度降低了约90%。该项目将继续与布里斯托大学、AHDB、Limagrain、RAGT、Saaten Union和先正达合作，对这些超低天冬酰胺小麦品系进行田间试验，以期为英国市场开发低丙烯酰胺小麦品种。在第一年，我们将为试验增加种子。我们还将研究CRISPR系的发芽率：到目前为止，它们的发芽率很差，但这可以通过低浓度的天冬酰胺处理来逆转。我们将检查天冬酰胺和其他潜在治疗方法的有效性和实用性。我们亦会发展基因标记/工具，让育种者能将超低天冬酰胺的特性融入育种品系。最后，我们将准备一份风险评估，并提交一份进行田间试验的许可申请。在第2年，我们将进行田间试验，分析低天冬酰胺品系的出苗、物理特性、发育差异、组成、产量和其他农艺性状。我们还将进行温室实验，以确定低天冬酰胺品系是否以与正常小麦相同的方式对缺硫作出反应。该项目将代表作物基因编辑的一个里程碑，据我们所知，田间试验是英国和欧洲第一个基因编辑小麦，而AHDB和小麦育种者的参与将提供一个直接的商业化途径，如果植物在田间试验中表现良好。

The presence of acrylamide in popular foods has become one of the most difficult problems facing the food industry and its supply chain. Acrylamide is a contaminant that forms from an amino acid called asparagine in its free (non-protein) form and sugars such as glucose, fructose and maltose. The reaction occurs during frying, baking, roasting, toasting and high-temperature processing. Acrylamide is classified as probably cancer-causing in humans and affects development and fertility at high doses. Products made from wheat and other cereals are major sources of dietary acrylamide.Acrylamide in food is covered by regulation (EU) 2017/2158 (2018), which set Benchmark Levels for the presence of acrylamide in food, described compulsory mitigation measures and required all food businesses to monitor the levels of acrylamide in their products. Methods developed to limit acrylamide formation during processing do not work for all products, may adversely affect product quality and are expensive to implement. Our approach has been to lower the acrylamide-forming potential of crop products: for wheat and other cereals that means reducing the concentration of free asparagine in the grain. The project arises from previous BBSRC-funded work in which we have demonstrated large varietal differences in free asparagine concentration in wheat grain and shown it to be highly responsive to sulphur deficiency and other nutritional and environmental stress factors, as well as disease. Asparagine is made by an enzyme called asparagine synthetase and wheat has five genes that encode this enzyme. One of these genes, TaASN2, has emerged as a genetic target because it is highly active specifically in the grain. In our current work, involving teams at Rothamsted Research, Univ. of Bristol and partners AHDB, KWS, Limagrain, RAGT, Saaten Union and Syngenta, we have used the genome editing technique, CRISPR-Cas9, to knock out TaASN2 genes. In addition, we have screened a wheat population in which mutations have been introduced by chemical mutagenesis (a much older technique that has been used in plant breeding since the 1950s) and identified lines with mutations in each of the six versions of TaASN2 in the wheat genome. The TaASN2 mutations in these lines are being 'stacked' by one of our partners and partial knockouts will be available by Year 2 of the project. The concentration of free asparagine in one of the CRISPR lines is reduced by approximately 90%. The aim of this project, which will continue the partnership with Univ. of Bristol, AHDB, Limagrain, RAGT, Saaten Union and Syngenta, is to undertake field trials of these ultra-low asparagine wheat lines with a view to the development of low acrylamide wheat varieties for the UK market. In year 1 we will bulk up seed for the trial. We will also investigate the germination rate of the CRISPR lines: they have shown poor germination so far but this can be reversed by treatment with low concentrations of asparagine. We will check the efficacy and practicality of the asparagine and other potential treatments. We will also develop genetic markers/tools to enable breeders to integrate the ultra-low asparagine trait into breeding lines. Lastly, we will prepare a risk assessment and submit an application for permission to hold the field trial. In year 2 we will conduct the field trial, analysing the low asparagine lines for emergence, physical characteristics, developmental differences, composition, yield and other agronomic characteristics. We will also conduct glasshouse experiments to ascertain whether the low asparagine lines respond to sulphur deficiency in the same way as normal wheat. The project would represent a landmark for crop gene editing, with the field trial, to our knowledge, being the first for gene edited wheat in the UK and Europe, while the involvement of AHDB and wheat breeders will provide a direct pathway to commercialisation if the plants perform well in the field trial.