剑麻斑马纹病菌EST-SSR标记开发与评价

摘要

Zebra spot disease caused by Phytophora nicotianae var.parasitica (or Phytophora parasitica var.nicotianae) is the most serious and devastating fungi disease in sisal.In the present study,a total of 5 519 non-redundant P.nicotianae were generated from the 10 525 publicly available EST sequences by CAP3 software,which used to mine potential microsatellites by microsatellite identification tool (MISA).A total of 199 SRR was found in P.nicotianae expressed-squence tags data.Of the 199 SSR,the most abundant SSR was the tri-nucleotide type,with the SSR numbers being 55,accounting for 27.6％,followed by the di-nucleotide and mono-nucleotide type,with the SSR numbers being 51 and 47,accounting for 25.6％ and 23.6％.Among the di-nucleotide repeats,AG/CT was the most motifs and accounted for 76.5％.The AAG/CTT and AGC/CTG was the most motifs in tri-nucleotide,being 15 and 11,accounting for 27.3％ and 20.0％,respectively.Further screening to the number of repetitions and motifs relatively large number of SSR among the 199 SRR,which the right position to develop molecular markers,and a total of 22 primers were developed.Among the 22 primer pairs designed and synthesized,there were only 9 primer pairs that amplified characteristic SSR bands in genomic DNA of 18 tested isolates of P.nicotiana from sisal,thus the transferability was 40.9％.The results showed that it is feasible for molecular markers using this method to develop the sisal zebra germs,only a transfer rate of problems.%剑麻斑马纹病是剑麻生产中具有破坏性的一种真菌病害.本研究从GenBank(http://www.ncbi.nlm.nih.gov/nucest)中随机下载10 525条寄生疫霉EST序列,经过去冗余处理后得到5 519条无冗余EST.利用MISA(MIcroSAtellite identification tool)软件进行SSR发掘,从中共搜索到199个1～6碱基SSR,其中三核苷酸重复基元类型最多,共鉴定到55个,占SSR总数的27.6％;其次是二核苷酸重复基元类型和单核苷酸重复基元类型,分别鉴定到51和47个,各占所鉴定SSR总数的25.6％和23.6％.进一步分析表明,AG/CT二核苷酸重复基元类型为优势重复类型,占二核苷酸SSR总数的76.5％.而AAG/CTT和AGC/CTG 2个基元类型在三核苷酸中出现频率最多,分别为15和11次,分别占三核苷酸SSR总数的27.3％及20.0％.从鉴定的199个SSR中,选取含有SSR的合适区段设计了22对引物,并通过18个剑麻斑马纹病菌菌株的基因组DNA进行了评价,结果只有其中9对引物能从剑麻斑马纹病菌基因组DNA中有效扩增,其扩增移效率为40.9％.由此表明,利用此方法来开发剑麻斑马纹病菌的分子标记具有可行性,只是存在一个转移效率问题.