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

加拿大每年生产13.8万公顷（460万吨，农场价值10亿美元）的马铃薯，美国的产量是加拿大的五倍。目前加拿大种植的马铃薯品种超过200种。品种CF 1 **（注册名称保密）在加拿大和美国都有种植，对由细菌病原体疥疮链霉菌引起的疮痂病非常敏感。病原体感染幼嫩的块茎，引起结痂，通常使块茎变形。目前的管理方案无效，往往导致严重的产量 ** 损失; 2015年，仅CF 1品种的损失就达80万美元。遗传抗性是控制这种疾病的最好方法。然而，一些商业品种的R基因组成相当差，因此只有低 ** 至中等水平的抗性。此外，性不亲和限制了马铃薯育种。基于 ** 代谢组学和转录组学，Kushalappa实验室已经鉴定了几个抗马铃薯晚疫病和小麦赤霉病的R基因。这些R基因生物合成抗性相关的代谢产物，这些代谢产物沉积在细胞壁上以加强细胞壁，从而遏制病原菌的传播;这种机制对于不同的植物和病原菌是共同的，因此可以赋予多种抗病性。在这个提议中，这些R基因中的一些将在CF 1中进行测序，如果是多态的，最好的一个将用于基因组编辑。将建立愈伤组织产生和植株再生的方案。基于使用双生病毒载体的CRISPR-Cas 9系统，CF 1中突变的R基因片段将被功能性基因片段替换。将基于扩增子测序在再生植物中确认 ** 替换的基因。将气培法生产 ** 顺式基因CF 1和原始CF 1的微型块茎，将用疮痂 ** 病原体接种幼块茎，并评估疾病严重程度以确认增强的抗性。在这个项目期之后，将与卡文迪什农场合作开发一个 ** 精心设计的项目，以确定更多的多态性基因，并在CF 1中编辑它们，以获得高 ** 水平的多重抗病性，并提交给 ** NSERC-SPG申请资金。

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.