Standardization of protocols for plant regeneration and genome editing CF1 potato cultivar to enhance scab resistance

加拿大的马铃薯产量为13.8万公顷（460万吨，每年农场价值10亿美元），美国的产量是加拿大的5倍。加拿大目前种植了200多种马铃薯品种。CF1**品种（注册名称保密）在加拿大和美国都有种植，非常容易感染疥疮**，这是由一种细菌病原体——疥疮链霉菌引起的。病原菌感染幼嫩的块茎，造成结痂性损伤，常常使块茎变形。目前的管理方案并不有效，经常导致严重的产量损失；2015年，仅CF1品种的损失为80万美元。遗传抗性是控制这种疾病的最好方法。然而，一些商品品种的R基因组成相当差，因此只有低**至中等水平的抗性。此外，性不亲和性限制了马铃薯的育种。基于代谢组学和转录组学，Kushalappa实验室已经鉴定出几个抗马铃薯晚疫病和小麦枯萎病的R基因。这些R基因生物合成与抗性相关的代谢物，这些代谢物沉积在细胞壁上，以抑制病原体的传播；这种机制在不同的植物和病原体中是共同的，因此可以赋予多重抗病能力。在本提案中，这些R基因中的一些将在CF1中进行测序，如果多态性，则将最好的一个用于基因组编辑。建立愈伤组织的产生和植株再生方案。基于双病毒载体crispr - cas9系统，将CF1中发生突变的R基因片段**替换为功能基因片段。**替换的基因将在再生植株中通过扩增子测序得到确认。将**基因CF1和原CF1的微型块茎进行气栽生产，接种痂**病原菌接种幼块茎，并评估疾病严重程度以确认抗性增强。本项目结束后，将与卡文迪什农场合作，开发一个详细的项目，以确定更多的多态性基因，并在CF1中对其进行编辑，以获得高水平的多种疾病抗性，并提交给NSERC-SPG申请资助。

Canada produces potato in 138 thousand ha (4.6 million t, farm gate value of $1 billion per annum) and US**produces five times more. More than 200 potato cultivars are currently grown in Canada. The cultivar CF1**(registered name held confidential) is grown both in Canada and also in USA and is very susceptible to scab,**caused by Streptomyces scabies, a bacterial pathogen. The pathogen infects young tubers and causes scabby**lesions, often deforming the tubers. Current management options are not effective, often leading to severe yield**losses; in 2015, the loss only for CF1 cultivar was $800 000. Genetic resistance is the best way to manage this**disease. However, the R gene compositions of several commercial cultivars are rather poor, thus have only low**to moderate levels of resistance. Furthermore, the sexual incompatibility limits potato breeding. Based on**metabolomics and transcriptomics Kushalappa lab has identified several R genes against late blight in potato**and to fusarium head blight in wheat. These R genes biosynthesize resistance related metabolites that are**deposited to reinforce the cell walls to contain the pathogen spread; this mechanism is common to different**plants and pathogens, thus can confer multiple disease resistance. In this proposal, some of these R genes will**be sequenced in CF1, and if polymorphic, the best one will be used in genome editing. The protocol for calli**production and plant regeneration will be established. The R gene segment that is mutated in CF1 will be**replaced with the functional gene segment, based on CRISPR-Cas 9 system using geminivirus vectors. The**replaced genes will be confirmed in the regenerated plants based on amplicon sequencing. The mini-tubers of**cisgenic CF1 and the original CF1 will be produced aeroponically, young tubers will be inoculated with scab**pathogen and disease severity will be assessed to confirm enhanced resistance. After this project period, an**elaborate project will be developed to identify more polymorphic genes and to edit them in CF1 to obtain high**levels of multiple disease resistance, in collaboration with the Cavendish Farms, and submitted to**NSERC-SPG for funding.