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Transcriptome Analysis of Resistance Mechanism to Potato Wart Disease

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Transcriptome Analysis of Resistance Mechanism to Potato Wart Disease Open Life Sciences 2021; 16: 475–481 Research Article Peihua Li, Ruihong Fan, Zhengsong Peng*, Yuan Qing, Zhirong Fang Transcriptome analysis of resistance mechanism to potato wart disease https://doi.org/10.1515/biol-2021-0045 received September 24, 2020; accepted December 18, 2020 1 Introduction Abstract: To understand the molecular mechanism of the Hailed as the “second apple” and the “second bread,” resistance to potato wart disease, we used the potato potato is the fourth biggest food crop worldwide after cultivar Qingshu 9 as the experimental material and pre- wheat, rice, and maize [1]. In China, the planting area pared potato samples with different levels of disease of potatoes reaches 5.3333 million hm2, only after that through inoculation. The RNAs of the samples were of wheat, maize, and rice. The annual output of potatoes extracted, and transcriptome analysis was performed on in China stands at 100 million tons, more than anywhere ( ) the samples not infected by the disease control group else [2]. As China strives to make potato a staple food, the ff and also on the samples with di erent levels of disease, high and stable output of potatoes is increasingly impor- - with the aid of high throughput sequencing. Next, the tant to food security in the country [3]. ff ( ) di erentially expressed genes DEGs associated with Potato wart disease, also called potato canker and fi the resistance to potato wart disease were identi ed black wart of potato, is a devastating soil-borne disease based on the analysis results. Using bioinformatic tools, widely distributed in cold, rainy mountain areas [4,5]. fi the DEGs were functionally annotated, classi ed, and The disease is caused by Synchytrium endobioticum (Schilb.) enriched in related metabolic pathways. The main results Percival, a soil-borne obligate parasitic fungus. Belonging are as follows: Compared with the control group, 4 DEGs to Synchytrium, Synchytriaceae, Chytridiales,andChytridio- were identified in the samples with light disease, 52 were mycota, the fungus has no mycelium and cannot be artifi- found in the samples with medium disease, and 214 were cially cultivated yet. discovered in the samples with heavy disease. Potato The pathogenic fungus, i.e., S. endobioticum (Schilb.) mainly resists the wart disease by suppressing its gene Percival, mainly damages the underground parts of pota- expression, and the degree of suppression depends on toes. The fungus causes the host cells to continuously the level of the disease; the disease resistance might be divide, forming large and small cauliflower-shaped tumors dominated by cellular nucleic acid-binding protein, AP2- and chapped skins on tuber or stolon. The canker tissues like transcription factor, and E3 ubiquitin-protein ligase. are yellowish white in the early stage. With the elapse of This research provides new insights into the molecular time, these tissues become dark brown, soft, and highly mechanism of potato resistance against wart disease. putrescible, giving off afoulodor[6–8]. Keywords: potato wart disease, food safety, high- Potato cancer was first discovered in Hungary in throughput sequencing, differentially expressed genes 1896. With the expansion of planting scale and the inflow of foreign potato varieties, it gradually spread to Central and Northern European countries. It was introduced in North America in 1912 and successively in South America, India, New Zealand, and other places. Now it has spread to about 50 countries on the five continents of the world. The dormant sporangia of the fungus exist in tubers * Corresponding author: Zhengsong Peng, College of Agronomy, infected with wart disease and the tiny soil particles on Xichang Univeristy, Xichang, Sichuan 615013, China, the tuber surface. After the tubers rot, the dormant spor- - e mail: [email protected] angia are released into the soil. These dormant sporangia Peihua Li, Yuan Qing, Zhirong Fang: College of Agronomy, [ – ] Xichang Univeristy, Xichang, Sichuan 615013, China can survive in the soil for more than 30 years 9 11 . Ruihong Fan: College of Agronomy, Sichuan Agricultural University, Despite the huge damage caused by the fungus, still no Chengdu, Sichuan 611130, China way has been found to effectively prevent S. endobioticum Open Access. © 2021 Peihua Li et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International License. 476 Peihua Li et al. (Schilb.) Percival from infecting potatoes, other than were subjected to RNA extraction, library construction and breeding for disease resistance. sequencing, and preliminary screening of key genes Two defining features of potato wart disease are serious involved in the resistance to the disease. The research damage and high-potential threat. On the one hand, the results provide new insights into the resistance mechanism disease directly affects the output and quality of potatoes of potato wart disease. in the field and causes potatoes to rot in the late harvest and storage period. The inflected potatoes are of poor quality and inedible, resulting in dull sale and major economic losses. On the other hand, it is difficult to remove the patho- 2 Materials and methods genic fungus that contaminates the soil in the short-term. Over the years, various chemical and biological measures 2.1 Materials have been tried to clean the soil, but to no avail [4]. Therefore, many countries have treated the wart dis- Our experiment uses the potato cultivar Qingshu 9 (selec- ease as a serious threat and quarantine infected objects in tion and breeding of Qinghai Academy of Agricultural potato production. The disease is characterized by high Sciences, China), which was provided by Sichuan Potato incidence and fast spread. On average, the output of plots Key Laboratory, Xichang University. is reduced by 20–30%. In severe cases, the disease could reduce the output by 80% and even cause total crop failure [12]. This calls for in-depth research on the patho- genic mechanism of potato wart disease. The research 2.2 Experimental method results help identifying the genes related to the disease, breeding disease-resistant varieties, and ensuring high 2.2.1 Sample preparation and stable output and also high-quality potatoes. In the research work of potato cancer-resistance gene To identify the differentially expressed genes (DEGs) of the location, Brugmans et al. used the densification of ampli- potato infected with the wart disease, three noninfected fied fragment length polymorphism (AFLP) markers and potatoes were allocated to the control group (L0),andthe the use of bacterial artificial chromosome (BAC) capture other potatoes were inoculated with the wart disease of and sequencing technology, and Ballvaora et al. used one seven different levels (L1–L7) by the Glynne–Lemmerzahl highly resistant and two different highly susceptible vari- method [6]. The samples with all eight levels of disease are eties of tetraploid potatoes to hybridize. Then molecular showninFigure1. marker methods such as simple sequence repeat (SSR), The inoculation was implemented in the following inter simple sequence repeat (ISSR), and random ampli- steps: Apply a 1- to 2-mm-thick warm Vaseline on the eyes fied polymorphic DNA (RAPD) were used on the F1 popu- of tubers, creating a water-retaining ring. Then drop steri- lation. Obidiegwu et al. used the F1 population obtained lized distilled water over the eyes. Place disease-causing by crossing the tetraploid potato high-resistant variety canker tissues, which has been developed for 3–4weeks, and the high-susceptible variety and used SSR markers on the eyes, and cover them up to avoid infection. After and single-nucleotide polymorphism (SNP) chips to ana- incubating at 10 ℃ for 48 h, remove the inoculum, remove lyze. The transcriptome is the sum of all RNAs transcribed Vaseline, and cover the samples with moist sterile soil or by a specific tissue or cell at a developmental stage or peat. Then cultivate the samples at 15–17℃ in the dark for functional state, including messenger RNAs (mRNAs), 25–28 days. Observe the growth and cancerization of the small RNAs, and noncoding RNAs (ncRNAs). Relying on samples every other day under the dissecting microscope. high-throughput sequencing (HTS),transcriptomesequen- cing determines mRNAs, small RNAs, or ncRNA sequences and quickly obtains the expression level of almost all tran- 2.2.2 Transcriptome sequences scripts of an organ or a tissue of a species under a certain state [13,14]. At present, transcriptome sequencing has been The total RNAs of potato samples (L0–L7) in Figure 1 were widely applied to major food crops. However, there is no extracted and purified with BioFit plant total RNA purification report on its application on potato wart disease. kit (http://www.biofit.com), where L0 is the control group, L1 In this research, the potato cultivar Qingshu 9 was and L2 are infected with light disease (light),L4andL5are taken as the material. Potato samples with different levels infected with medium disease (medium),andL6andL7are of disease were obtained through inoculation. The samples infected with heavy disease (heavy). Transcriptome analysis of resistance mechanism to potato wart disease 477 Figure 1: Potato samples with different levels of wart disease. After passing the test, the RNA samples were sent respectively, using KafamKOALA (KEGGs)[15], AgriGO to BioMarker for library construction and transcriptome 2.0 [16], and omicshare software (https://www.omicshare. analysis by standard methods. The sequencing depth com/). was 6G per sample. The samples in each group were sequenced twice. 3 Results 2.2.3 Analysis of transcriptome data Transcriptome analysis showed that seven groups (L0, The transcriptome data were completely analyzed by CLC L1, L2, L4, L5, L6, and L7) were consistent in the number Genomics Workbench 10. For the reads of the original of original reads and the mean length (Table S1). After the sequences obtained through sequencing, the Trim reads unsuitable reads were eliminated by Trim reads program, program was called to remove the reads with an N (the eight groups had a slight difference in the number of base information that cannot be determined) proportion high-quality reads and the mean length.
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