Pathogens rely on expression of host susceptibility (S) genes to promote infection and disease. As DNA methylation is an epigenetic modification that affects gene expression, blocking access to S genes through targeted methylation could increase disease resistance. Xanthomonas phaseoli pv. manihotis, the causal agent of cassava bacterial blight (CBB), uses transcription activator-like20 (TAL20) to induce expression of the S gene MeSWEET10a. In this work, we direct methylation to the TAL20 effector binding element within the MeSWEET10a promoter using a synthetic zinc-finger DNA binding domain fused to a component of the RNA-directed DNA methylation pathway. We demonstrate that this methylation prevents TAL20 binding, blocks transcriptional activation of MeSWEET10a in vivo and that these plants display decreased CBB symptoms while maintaining normal growth and development. This work therefore presents an epigenome editing approach useful for crop improvement.

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ADDITIONAL INDEX WORDS. Blosyrus asellus, Cylas formicarius elegantulus, Ipomoea batatas var. batatas, rough sweetpotato weevil, tissue culture SUMMARY. Tissue-cultured, virus-tested (TC) plantlets of sweetpotato (Ipomoea batatas var. batatas) cultivars Okinawan, LA 08-21p, and Murasaki-29 were obtained from Louisiana State University Agricultural Center. The objectives of field trials conducted at the Kula Agricultural Park, Maui, HI, were to compare yield and pest resistance of 1) 'Okinawan' obtained from a commercial (C) field with TC 'Okinawan' and 2) TC Okinawan with the aforementioned TC cultivars. Trials were planted Oct. 2015 and Aug. 2016 and harvested 5 months later. Storage roots were graded according to State of Hawai'i standards, and marketable yields included Grades AA, A, and B. In addition, injuries due to sweetpotato weevil (Cylas formicarius elegantulus) or rough sweetpotato weevil (Blosyrus asellus) were estimated. In both trials, fresh and dry weights of marketable storage roots of TC 'Okinawan' were nearly twice those from commercial planting material. In both trials, marketable fresh weights differed among the three TC cultivars; however, significant interactions were found, indicating that yields of cultivars differed between years. In the first field trial, 'LA 08-21p' had fresh marketable yields 1.6 to 1.7 times greater than TC 'Okinawan' and Murasaki-29, respectively. In the second trial, fresh marketable yields of TC 'Okinawan' and 'LA 08-21p'were similar and 1.7 to 1.5 times greater than that of 'Murasaki-29', respectively. In both trials, 'LA 08-21p' had greater sweetpotato weevil injury than did the other two cultivars. Interestingly, in the second year, TC 'Okinawan' had greater rough sweetpotato weevil injury than did the other cultivars. Our results indicate that tissue-cultured planting materials increased marketable yields of TC 'Okinawan' compared with C 'Okinawan' sweetpotato and that the other TC cultivars did not produce greater yields than TC Okinawan.

Provitamin A biofortification and increased dry matter content are important breeding targets in cassava improvement programs worldwide. Biofortified varieties contribute to the alleviation of provitamin A deficiency, a leading cause of preventable blindness common among pre-school children and pregnant women in developing countries particularly Africa. Dry matter content is a major component of dry yield and thus underlies overall variety performance and acceptability by growers, processors, and consumers. Single nucleotide polymorphism (SNP) markers linked to these traits have recently been discovered through several genome-wide association studies but have not been deployed for routine marker-assisted selection (MAS). This is due to the lack of useful information on markers’ performances in diverse genetic backgrounds. To overcome this bottleneck, technical and biological validation of the loci associated with increased carotenoid content and dry matter content were carried out using populations independent of the marker discovery population. In the present study, seven previously identified markers for these traits were converted to a robust set of uniplex allele-specific polymerase chain reaction (PCR) assays and validated in two independent pre-breeding and breeding populations. These assays were efficient in discriminating marker genotypic classes and had an average call rate greater than 98%. A high correlation was observed between the predicted and observed carotenoid content as inferred by root yellowness intensity in the breeding (r = 0.92) and pre-breeding (r = 0.95) populations. On the other hand, dry matter content-markers had moderately low predictive accuracy in both populations (r< 0.40) due to the more quantitative nature of the trait. This work confirmed the markers’ effectiveness in multiple backgrounds, therefore, further strengthening their value in cassava biofortification to ensure nutritional security as well as dry matter content productivity. Our study provides a framework to guide future marker validation, thus leading to the more routine use of markers in MAS in cassava improvement programs.