金针菇rDNA序列结构特征分析

Sequence analyses of the rDNA structure of Flammulina filiformis

rDNA序列中的ITS作为DNA barcoding广泛应用于真菌的系统发育与物种辅助鉴定,IGS被认为可以用于种内水平不同菌株的鉴别。食用菌中还没有完整的rDNA序列的报道。本研究采用二代和三代测序技术分别对金针菇单核菌株"6-3"进行测序,用二代测序的数据对三代测序组装得到的基因组序列进行修正,得到一个在基因完整性、连续性和准确性均较好的基因组序列,对比Fibroporia vaillantii rDNA序列,获得金针菇完整的rDNA序列。金针菇rDNA序列结构分析表明,它有8个rDNA转录单元,长度均为5 903bp,有9个基因间隔区,其长度有较大差异,3 909–4 566bp。rDNA转录单元中,各元件的序列长度分别为:18S rDNA 1 796bp、ITS1 234bp、5.8S rDNA 173bp、ITS2 291bp、28S rDNA 3 410bp。基因间间隔区中,IGS1 1 351–1 399bp、5S rDNA 124bp、IGS2 2 435–3 092bp。金针菇的5S、5.8S、18S、28S rDNA序列准确性得到转录组数据的验证,也得到系统发育分析结果的支持。多序列比对发现,不同拷贝的基因间间隔区序列(IGS1和IGS2)存在丰富的多态性,多态性来源于SNP、InDel和TRS(串联重复序列),而TRS来源于重复单元的类型和数量。9个基因间间隔区之间,IGS1只有少量的SNP和InDel,IGS2不仅有SNP和InDel,还有TRS。本研究结果提示,在应用IGS进行种内水平不同菌株之间的鉴别时,需要选取不同拷贝之间的保守IGS序列。

The ITS in rDNA sequence is widely used as DNA barcoding in the study of phylogentic development of fungi and identification of species.IGS could be used for identification of different strains in different species.No complete rDNA sequence has been reported in edible fungi.In this study,next‐generation sequencing(NGS)and third generation sequencing(TGS)techniques were used to sequence a single monokaryon strain “6‐3” of Flammulina filiformis,and the genome sequences obtained by TGS were modified by NGS data to obtain a genome sequence with better gene integrity,continuity and accuracy.In comparison with Fibroporia vaillantii rDNA sequence,the complete rDNA sequence of Flammulina filiformis was obtained.rDNA sequence analysis showed that Flammulina filiformis had eight rDNA transcriptional units with a length of 5 903bp and 9 gene spacers with considerable difference in length ranging from 3 909 to 4 566bp.In the rDNA transcription unit,the sequence length of each element are: 18S rDNA 1 796bp,ITS1 234bp,5.8S rDNA 173bp,ITS2 291bp,and 28S rDNA 3 410bp.In intergenic spacers,IGS1 1 351–1 399bp,5S rDNA 124bp,and IGS2 2 435–3 092bp.The accuracy of 5S,5.8S,18S and 28S rDNA sequences of Flammulina filiformis was verified by transcriptome data and supported by phylogenetic analysis.Multiple sequence alignment indicated that there were abundant polymorphisms in the intergenic spacer sequences(IGS1 and IGS2)of different copies.Polymorphism was derived from SNP,InDel and TRS(tandem repeat sequences),and TRS was derived from the type and number of repeating units.Among the nine intergenic spacers,IGS1 has only a small number of SNP and InDel,while IGS2 not only has SNP and InDel,but also TRS.The results suggest that conservative IGS sequences between different copies should be selected when IGS is used to identify different strains at intraspecific level.