黄渤海沉积物真菌分子多样性及新种鉴定

发布时间：2018-06-11 15:43

本文选题：分子系统发生 + 焦磷酸测序　；参考：《中国海洋大学》2014年硕士论文

【摘要】：本研究对2013年6月采集自黄渤海30个站位的沉积物样品提取宏基因组DNA，利用454焦磷酸测序技术对ITS1和ITS2两个分子标记进行高通量测序，对有效序列依据97%相似度水平划分OTU。研究真菌在不同海域和不同分类学水平上的多样性和群落结构。测定沉积物环境理化因子，包括总碳（TOC）、总氮（TN）、C/N、总磷（TP）、总硫（TS）、总钾（TK）、pH和平均粒径。通过偏相关分析，研究真菌多样性与环境因子的关系。通过真菌群落与环境因子的冗余分析，识别影响黄渤海沉积物真菌群落结构的主要环境因子。 ITS1焦磷酸测序获得有效序列122,211条，76.98%的序列可鉴定到属，共发现3,714个OTU，290个种系型，属于257个属。ITS2焦磷酸测序获得有效序列119,776条，87.91%的序列可鉴定到属，共发现6,676个OTU，232个种系型，属于201个属。南黄海、北黄海和渤海三大海域之间的OTU与种系型数量均无显著性差异。在目水平上比较不同海域真菌平均OTU数量差异。ITS1的分析结果表明，南黄海真菌OTU数量极显著高于北黄海和渤海，北黄海极显著高于渤海；而ITS2的分析结果则表明，南黄海极显著低于北黄海和渤海，北黄海与渤海无差异。在门水平上比较不同海域同一类群真菌OTU数量差异。黄渤海沉积物子囊菌OTU数量无显著差异。ITS1分析认为南黄海担子菌OTU数量显著大于渤海，而ITS2分析结果与之相反。ITS1分析认为球囊菌OTU数量无显著差异，而ITS2分析认为渤海和北黄海的球囊菌OTU显著多于南黄海。 ITS1空间分布型分析结果表明，南黄海和北黄海沉积物真菌OTU呈聚集分布，而渤海沉积物真菌呈随机分布。ITS2的空间分布型结果表明，，北黄海沉积物真菌OTU呈聚集分布，南黄海和渤海真菌OTU呈随机分布；ITS1和ITS2空间分布型结果均表明，三大海域沉积物真菌种系型均呈随机分布。三大海域沉积物真菌Shannon-Wiener多样性指数无显著差异。子囊菌门粪壳菌纲和座囊菌纲、担子菌门伞菌纲为优势类群。同一海域的沉积物样品具有相似的真菌群落结构。调查范围内，纬度由低到高，各海域真菌多样性大致呈现由高到低的梯度变化，即南黄海、北黄海、渤海的真菌群落组成复杂度依次降低。影响黄渤海沉积物真菌群落组成的最重要的环境因子为TN、C/N、pH。尽管以ITS1和ITS2作为分子标记，在高级分类介元上能得到类似的分类学结果，但后续多样性和群落结构分析存在一定程度的差异，PCR扩增的偏好性是其中最主要的原因。ITS1F/ITS2因其良好的真菌特异性，因而可以扩增得到更多的真菌序列；其片段长度更长，信息位点更多，且平均变异程度更高。本研究结果也表明，在分析真菌群落结构和多样性时，ITS1优于ITS2。随着测序成本的不断降低，同时采用两个片段进行测序分析，互为补充，可能得到更加准确的结果。同时，应考虑singletons和doubletons的处理方法，以减少偶见种对多样性分析的影响。此外，本研究采用形态学和分子系统学相结合的方法，对分离自黄海山东沿岸潮间带海藻（内生真菌）或沉积物的疑难真菌进行形态学描述和系统发育分析，鉴定出三个新种Phaeoisaria sedimenticola、Ascotricha longipila、Ascotrichaparvispora和一个新组合种Ascotricha sinuosa。除Phaeoisaria sedimenticola为无性型外，其余均为全型。通过探讨刺囊壳属（Ascotricha）的系统发育地位及其无性型，明确了刺囊壳属应归于炭角菌科（Xylariaceae）炭角型类群（Xylarioideae），其无性型是双聚散霉属（Dicyma），汉斯霉属（Hansfordia）可能与刺囊壳属有关。
[Abstract]:In this study, the macrogenome DNA was extracted from the sediment samples collected from 30 stations from yellow and Bohai in June 2013. The 454 pyrophosphoric acid sequencing technology was used to sequence the high flux sequence of two molecular markers of ITS1 and ITS2, and the effective sequences were divided by 97% similarity levels to study the diversity and group of fungi at different sea regions and different taxonomic levels. Determination of sediment environmental physical and chemical factors, including total carbon (TOC), total nitrogen (TN), C/N, total phosphorus (TP), total sulfur (TS), total potassium (TK), pH and average particle size. Through partial correlation analysis, the relationship between fungal diversity and environmental factors was studied. By the redundancy analysis of fungal communities and environmental factors, the main factors affecting the community structure of the sediments in the yellow and Bohai sediments were identified. Environmental factors.
ITS1 pyrophosphate sequencing obtained 122211 effective sequences, 76.98% sequences can be identified, a total of 3714 OTU, 290 species, 257 genera.ITS2 pyrophosphoric sequence to obtain an effective sequence of 119776, 87.91% of the sequence can be identified, a total of 6676 OTU, 232 species, 201 genera, South Yellow Sea, north the Yellow Sea and Bohai three. There was no significant difference in OTU between the sea area and the number of species.
The analysis of the average OTU number difference.ITS1 of fungi in different sea areas showed that the number of OTU in the Southern Yellow Sea was significantly higher than that in northern the Yellow Sea and Bohai, and the northern the Yellow Sea was significantly higher than that in Bohai, while the ITS2 analysis showed that the Southern Yellow Sea was significantly lower than that of northern the Yellow Sea and Bohai, and the north the Yellow Sea and Bohai were not different.
The number of OTU in the same group of fungi in the different sea areas was compared. There was no significant difference in the number of OTU in the Yellow Bohai sediments. The number of OTU in the Southern Yellow Sea basidiomycetes was significantly greater than that in Bohai, while the ITS2 analysis and the opposite.ITS1 analysis showed that there was no significant difference in the number of OTU in the balloon, while ITS2 analysis considered Bohai and North Yellow. The OTU of the sea was significantly more than the South Yellow Sea.
The results of ITS1 spatial distribution analysis showed that the OTU of sediment fungi in the Southern Yellow Sea and north the Yellow Sea was distributed, and the spatial distribution pattern of the random distribution of.ITS2 in the Bohai sediment fungi showed that the OTU of the sediments in the northern the Yellow Sea was clustered, and the OTU of the Southern Yellow Sea and Bohai fungi was distributed randomly, and the spatial distribution of ITS1 and ITS2 showed that the fungi were distributed randomly. The species of fungal species of sediment in the three sea areas were distributed randomly. There was no significant difference in the Shannon-Wiener diversity index of the sediment fungi in the three sea areas.
The sediment samples in the same sea area have similar fungal community structure. In the range of investigation, the latitude from low to high, the diversity of fungal communities in each sea area generally presents a high to low gradient, that is, the complexity of the fungal community in the Southern Yellow Sea, north the Yellow Sea and Bohai decreases in turn. The most important environment affecting the composition of the yellow and Bohai sediment fungi community is in turn. Factors are TN, C/N, pH.
Although ITS1 and ITS2 are used as molecular markers, similar taxonomic results can be obtained on the high level taxonomy, but there are some differences in subsequent diversity and community structure analysis. The preference of PCR amplification is the most important reason for its good fungal specificity, so that more fungal sequences can be amplified. The results also showed that ITS1 was better than ITS2. in analyzing fungal community structure and diversity, and that the two fragments were sequenced and filled with more accurate results. At the same time, sing should be considered. Letons and doubletons were used to reduce the effect of occasional species on diversity analysis.
In addition, the morphological and molecular phylogeny method was used to describe the morphological description and phylogenetic analysis of the difficult fungi isolated from the intertidal seaweeds (endophytic fungi) or sediments from the coast of Shandong, the Yellow Sea, the Yellow Sea, and identified three new species of new species, sedimenticola, Ascotricha longipila, Ascotrichaparvispora and a new species. The combination of Ascotricha sinuosa., except Phaeoisaria sedimenticola, is an asexual type, and the rest are all type. By discussing the phylogenetic status and asexual type of the genus Ascotricha, it is clear that the genus acanthopanacis should be attributed to the carbon angle group (Xylarioideae) of the genus Xylariaceae (Xylariaceae), and its asexual type is double polymildew (Dicyma) and Hans mold. The genus (Hansfordia) may be related to the genus spacaclis.
【学位授予单位】：中国海洋大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：Q178.53

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