克山病病区自产玉米霉变真菌多样性初步检测报告

A pilot study of fungi diversity in moldy corns grown in Keshan disease areas

目的检测克山病病区霉变玉米真菌谱带，获得其中优势菌群的信息，为后续次生毒素研究提供依据。方法在克山病病区（甘肃省庆阳市合水县、正宁县，黑龙江省尚志市元宝镇）及非病区（黑龙江省哈尔滨市郊区）4个采样点，每个采样点采用简单随机抽样的方法采集当地种植的新脱粒玉米样品5份，共20份，置于无菌自封袋内密封、编号并运回实验室。放置于真菌培养箱内，霉焐培养2周后，进行真菌总DNA的提取。然后将DNA样品送至北京奥维森基因科技有限公司进行质检，并利用高通量DNA测序技术及MiSeq PE3000平台对真菌内转录间隔区1（ITSl）进行测序分析，通过稀释性曲线及香农曲线是否趋于平缓分析测序数据量的合理性，通过物种组成覆盖率（Coverage）是否〉99％分析测试样品中真菌群落的真实情况，根据真菌物种在各组中的相对丰度寻找病区组与非病区组之间的差异菌属。结果高通量DNA测序分析共获得513869条优质序列，共产生528个操作分类单元（OUT）。稀释性曲线及香农曲线的趋势是逐渐趋于平缓的，表明本次测序数据量合理；Coverage值均〉99％，表明测序结果能反映样品中真菌群落的真实情况。物种组成分析表明。克山病病区和非病区间真菌种类组成有很多重叠，但Wickerhamomyces属、念珠菌属和曲霉菌属相对丰度在病区组高于非病区组。结论Wickerhamomyces属、念珠菌属、曲霉菌属是3个病区组中的优势菌，可能与克山病发病有关，可考虑用于后续次生毒素的研究。

Objective To carry out a pilot investigation into fungus diversity in corn samples harvested from Keshan disease areas and obtain the information of dominant fungi, to predict possible secondary toxins in future study. Methods In Keshan disease areas （Heshui County and Zhengning County of Gansu Province, Yuanbao Town of Heilongjiang Province） and non-Keshan disease area （Harbin suburbs of Heilongjiang Province）, 5 samples of newly harvested corn from local plants were collected via the simple random sampling method, and the samples （n = 20） were put in a sterile bags and sealed, numbered and transported to laboratory, placed in the incubator for cultivating mould fungi, cultured for 2 weeks, and fungal DNA was extracted. The DNA samples were sent to Beijing Auwegene Technology Co Ltd for quality inspection, and sequencing analysis of fungal internal transcribed spacer 1 （ITS1） region was done using modern high-throughput DNA sequencing technique and MiSeq PE3000 platform, the rationality of sequencing data was analyzed through the rarefaction curve and the Shannon-Wiener curve trend to be gentle or not, and the true condition of the fungal community in the sample was tested by species composition Coverage 〉 99% or not, the difference in fungal community was studied between Keshan disease group and non- Keshan disease group based on the relative abundance of the fungal species in each group. Results A total of 513 869 high quality sequences were obtained, and 528 operational taxonomic unit （OTU） classifications were produced. The trend of rarefaction curve and Shannon-Wiener curve were gradually flattening, they showed that the sequences of the data were reasonable; and coverage 〉 99% showed that data could reflect the true situation of the fungal community. Analysis of species composition showed that there was a great overlap of fungal species between Keshan disease group and non-Keshan disease group, the relative abundance of the genus Wickerhamomyces, Candida and Aspergillus in Keshan disease group were higher than that of non-Keshan disease group. Conclusion Genus Wickerhamomyces, Candida and Aspergillus may be the dominant fungi associated with Keshan disease, it can be considered for secondary toxins studies.