基于基因组de novo测序的丛赤壳科真菌交配型相关基因鉴定及其系统学应用

Ascomyctes are a large group with very high species diversity and a broad range of lifestyles in the Kingdom of Fungi and closely related to humans. With the development of high-throughput sequencing technologies, investigation of mating type (MAT) genes on the basis of fungal genomes is becoming possible and feasible. This proposal is to aim at some well-circumscribed species of Nectriaceae that their genomes were sequenced using Illumina-Solexa technology. The raw sequencing data were assembled using de novo sequence assembly software ABySS, SOAPdenovo, VELVET, and IDBA, and assemblies with the highest N50 values were selected for further analysis. .Available MAT genes in Sordariomycetes were obtained from GenBank and select representatives used in StandAlone BLAST. Other genes were obtained which are reported to be commonly found near MAT genes: DNA lyase APN2, cytoskeleton assembly control protein SLA2, anaphase-promoting complex protein APC5, cytochrome c oxidase subunit COX13, and pre-mRNA splicing factor CWF24. These genes were used as query sequences against a database containing the assemblies of sequenced genomes. BLAST software was used to pull out the contigs containing the target MAT loci for closer examination..The sequences of the detected mating type genes were used to design primers targetting ca 1 kb fragments for Nectriaceous fungi. These primers were tested on between 30 to 100 isolates for each species. Then, application of phylogenetic relationships within the family Nectriaceae was investigated using MAT genes as molecular markers to establish a more natural classification system. .This research is to investigate the locus and types of fungal mating type and associated genes and their application for phylogeny on the basis of genomic level. The results will not only provide useful data for comparative fungal genomics studies, but also be beneficial and useful for understanding fungal life history, conversion of reproduction, propagation mechanisms， and survival strategy.

子囊菌是真菌界物种丰富、生活史多样的类群，与人类关系密切。随着高通量测序技术的发展，在基因组范围研究真菌交配型（mating type, MAT）成为可能。本项目拟对丛赤壳科中分类地位明确的代表真菌进行基因组de novo测序并利用ABySS、SOAPdenovo、Velvet和IDBA等软件对原始序列数据进行组装；利用GenBank中粪壳菌纲真菌MAT及相关基因APN2、SLA2、APC5等作为查询序列，对丛赤壳科真菌基因组进行BLAST比对、预测其MAT基因类型及结构；通过引物设计、PCR和序列测定等分子生物学手段进行验证，探讨MAT基因在丛赤壳科属、种间的演化关系与系统学应用。本研究在基因组水平开展真菌MAT相关基因鉴定，深入探讨MAT基因位点、类型及其在系统学上的应用，其结果不仅为比较真菌基因组学研究提供数据，而且为认识真菌生活史、繁殖方式转换、传播机制和真菌生存策略提供科学依据。

丛赤壳科（Nectriaceae）隶属于子囊菌门粪壳菌纲肉座菌目，其成员世界性分布，主要为植物和其他真菌上的寄生、兼性寄生或腐生生物，其中包括一些重要的经济植物病原菌，还有一些可产生具有药用或生物防治价值的代谢产物，少数产生真菌毒素，是子囊菌中具有重要经济价值和生态重要性的类群。.随着高通量测序技术的发展，在基因组水平开展丛赤壳科真菌交配型基因鉴定及基因类型与结构研究，探讨交配型基因在系统学上的应用。本项目研究包括：①运用生物信息学手段对丛赤壳科菌株基因组de novo 测序数据进行组装、基因预测、基因注释及重复序列分析等，评估了ABySS、SOAP、Velvet、MaSuRCA和IDBA-UD的K-mer、N50等参数；②选择Fusarium proliferatum、F. verticillioides、Gibberella fujikuroi 等的MAT基因片段作为查询序列，对获得的丛赤壳科基因组进行BLASTN、TBLASTX 比对，预测该科真菌相应基因的同源序列，分析基因位点及结构；③通过引物设计、PCR、测序验证其MAT 基因，根据MAT 基因开展育性分析和有性型诱导研究；④基于MAT及相关基因的同源性设计特异性分子标记，探讨丛赤壳科各属、种间关系及该科与其他类群间亲缘关系。项目研究结果不仅为比较真菌基因组学研究提供数据，而且为认识真菌生活史、繁殖方式转换、传播机制和真菌生存策略提供科学依据。.项目实施期间，共采集标本600余份，分离纯培养菌株60余株, 其中，丛赤壳科标本50余份、菌株30株，发现新种和中国新记录种各1个。发表署名项目编号的研究论文6篇，10人次参加国内学术会议，2人次参加国际学术交流，先后培养硕士研究生13名和本科生多名，其中7名硕士研究生和1名本科生参与项目相关研究。