本文選題：分子系統(tǒng)發(fā)生 + 焦磷酸測序　； 參考：《中國海洋大學》2014年碩士論文

【摘要】：本研究對2013年6月采集自黃渤海30個站位的沉積物樣品提取宏基因組DNA，利用454焦磷酸測序技術(shù)對ITS1和ITS2兩個分子標記進行高通量測序，對有效序列依據(jù)97%相似度水平劃分OTU。研究真菌在不同海域和不同分類學水平上的多樣性和群落結(jié)構(gòu)。測定沉積物環(huán)境理化因子，包括總碳（TOC）、總氮（TN）、C/N、總磷（TP）、總硫（TS）、總鉀（TK）、pH和平均粒徑。通過偏相關(guān)分析，研究真菌多樣性與環(huán)境因子的關(guān)系。通過真菌群落與環(huán)境因子的冗余分析，識別影響黃渤海沉積物真菌群落結(jié)構(gòu)的主要環(huán)境因子。 ITS1焦磷酸測序獲得有效序列122,211條，76.98%的序列可鑒定到屬，共發(fā)現(xiàn)3,714個OTU，290個種系型，屬于257個屬。ITS2焦磷酸測序獲得有效序列119,776條，87.91%的序列可鑒定到屬，共發(fā)現(xiàn)6,676個OTU，232個種系型，屬于201個屬。南黃海、北黃海和渤海三大海域之間的OTU與種系型數(shù)量均無顯著性差異。 在目水平上比較不同海域真菌平均OTU數(shù)量差異。ITS1的分析結(jié)果表明，南黃海真菌OTU數(shù)量極顯著高于北黃海和渤海，北黃海極顯著高于渤海；而ITS2的分析結(jié)果則表明，南黃海極顯著低于北黃海和渤海，北黃海與渤海無差異。 在門水平上比較不同海域同一類群真菌OTU數(shù)量差異。黃渤海沉積物子囊菌OTU數(shù)量無顯著差異。ITS1分析認為南黃海擔子菌OTU數(shù)量顯著大于渤海，而ITS2分析結(jié)果與之相反。ITS1分析認為球囊菌OTU數(shù)量無顯著差異，而ITS2分析認為渤海和北黃海的球囊菌OTU顯著多于南黃海。 ITS1空間分布型分析結(jié)果表明，南黃海和北黃海沉積物真菌OTU呈聚集分布，而渤海沉積物真菌呈隨機分布。ITS2的空間分布型結(jié)果表明，，北黃海沉積物真菌OTU呈聚集分布，南黃海和渤海真菌OTU呈隨機分布；ITS1和ITS2空間分布型結(jié)果均表明，三大海域沉積物真菌種系型均呈隨機分布。三大海域沉積物真菌Shannon-Wiener多樣性指數(shù)無顯著差異。子囊菌門糞殼菌綱和座囊菌綱、擔子菌門傘菌綱為優(yōu)勢類群。 同一海域的沉積物樣品具有相似的真菌群落結(jié)構(gòu)。調(diào)查范圍內(nèi)，緯度由低到高，各海域真菌多樣性大致呈現(xiàn)由高到低的梯度變化，即南黃海、北黃海、渤海的真菌群落組成復雜度依次降低。影響黃渤海沉積物真菌群落組成的最重要的環(huán)境因子為TN、C/N、pH。 盡管以ITS1和ITS2作為分子標記，在高級分類介元上能得到類似的分類學結(jié)果，但后續(xù)多樣性和群落結(jié)構(gòu)分析存在一定程度的差異，PCR擴增的偏好性是其中最主要的原因。ITS1F/ITS2因其良好的真菌特異性，因而可以擴增得到更多的真菌序列；其片段長度更長，信息位點更多，且平均變異程度更高。本研究結(jié)果也表明，在分析真菌群落結(jié)構(gòu)和多樣性時，ITS1優(yōu)于ITS2。隨著測序成本的不斷降低，同時采用兩個片段進行測序分析，互為補充，可能得到更加準確的結(jié)果。同時，應(yīng)考慮singletons和doubletons的處理方法，以減少偶見種對多樣性分析的影響。 此外，本研究采用形態(tài)學和分子系統(tǒng)學相結(jié)合的方法，對分離自黃海山東沿岸潮間帶海藻（內(nèi)生真菌）或沉積物的疑難真菌進行形態(tài)學描述和系統(tǒng)發(fā)育分析，鑒定出三個新種Phaeoisaria sedimenticola、Ascotricha longipila、Ascotrichaparvispora和一個新組合種Ascotricha sinuosa。除Phaeoisaria sedimenticola為無性型外，其余均為全型。通過探討刺囊殼屬（Ascotricha）的系統(tǒng)發(fā)育地位及其無性型，明確了刺囊殼屬應(yīng)歸于炭角菌科（Xylariaceae）炭角型類群（Xylarioideae），其無性型是雙聚散霉屬（Dicyma），漢斯霉屬（Hansfordia）可能與刺囊殼屬有關(guān)。
[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.
【學位授予單位】：中國海洋大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：Q178.53

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相關(guān)期刊論文 前2條

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2 郭良棟;;中國微生物物種多樣性研究進展[J];生物多樣性;2012年05期

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