Cloning And Sequence Analysis Of The COI Gene In Botrytis Species And Related Fungi

Gray mold is an important worldwide disease.It is caused by Botrytis and Botrytis-like fungi and occurs on plants grown on bare fields as well as in greenhouse.This disease is difficult to control.Because morphological features of Botrytis and related fungi are very similar.It is difficult to distinguish Botrytis species and related fungi using traditional methods.In this study,47 strains of 19 species were collected from our laboratory,then cloning and analysising COI gene,to explored whether this gene can be used to classify and identify Botrytis species and related fungi.The results obtained are listed as followings:(1)The length of all exons of COI gene of 47 strains of 19 species is 1719 bp.They were predicted to encode polypeptides with 572 amino acids.Identity of exon sequences of COI in 47 strains was high,by 99.96%-100%within a species and by 98.08%-100%between species.Botrytis spp.were similar to Streptobotrys by 98.25%-99.53%and to Amphobotrys ricini by 96.28%-97.79%.Besides the certain difference between Amphobotrys and Botrytis,identity of all exons of other strains were higher than 98%,identity between Botrytis cinerea and Botrytis pseudocinerea was as high as 100%.Therefore,these exon sequences of COI cannot be used for taxonomy and identification of Botrytis and related fungi.(2)The intraspecific identity of complete sequence of the COI gene in 47 strains is very high,except 3 species were between 84%-89%,the rest species were above 99%.Interspecific identity is very low.The length of the COI gene is 9219-24348 bp long.They contain large introns.The number of the introns varied from 6 to 13.The intron insertion sites are stable.There were a total of 18 intron insertion sites in these COI DNA sequences.Among these introns,11 introns occurred frequently(more than 3 times),whereas 7 sites occurred less frequently(less than 3 times).The size of DNA of the introns have a great variation,ranging from 515 bp to3,152 bp.Distribution of these introns presented in patchiness.COI complete sequence differed greatly among the 19 species.The differences were mainly reflected on the introns.Therefore,the introns of COI can be used for classification of Botrytis and related fungi.(3)Three pairs of specific PCR primers were desighed.They were BPCF/BPCR,BF/BR and 7FW/7R.The primer pair BPCF/BPCR could be used to distinguish Botrytis cinerea from Botrytis pseudocinerea.The primer pair BF/BR could be used to distinguish Botrytis fabae from Botrytis fabiopsis.The primer pair 7FW/7R could be used to distinguish Botrytis spp.and Streptobotrys from the other investigated sclerotiniaceous fungi.