利用高通量测序检测新疆野苹果上的病毒

Virus detection of Malus sieversii from Xinjiang using high-throughput sequencing

新疆野苹果Malus sieversii(Ledeb.)Roem.是苹果的祖先,也是重要的育种遗传资源,常用作砧木。近年来,新疆野苹果的分布面积骤减,对其的保护越来越重要。本研究采用高通量测序技术,对采自新疆伊犁天山野果林的26份样本进行病毒检测,检测到苹果褪绿叶斑病毒(apple chlorotic leaf spot virus,ACLSV)和苹果茎痘病毒(apple stem pitting virus,ASPV)。通过RT-PCR检测所有样品(127份),检测到ACLSV、ASPV和苹果茎沟病毒(apple stem grooving virus,ASGV),检出率分别为22%、19.7%和11%。结合RACE PCR技术扩增得到ASGV新疆野苹果分离物基因组全长序列,暂时命名为ASGV-XJ。去除3′末端poly(A)后的长度为6506 bp,有两个彼此重叠的开放阅读框(open reading frame,ORF),ORF1(47-6364 nt)编码一个241 kD的多聚蛋白,包含甲基转移酶(methyltransferase)、木瓜蛋白酶(papain-like-protease)、解旋酶(nucleotide triphosphate-binding helicase)、RNA依赖的RNA聚合酶(RNA-dependent RNA polymerase)和C端的外壳蛋白(coat protein,CP)等;ORF2(4798-5760 nt)编码一个36 kD的运动蛋白(movement protein,MP)。系统发育分析表明,分离物间没有表现出寄主专一性和地理分布规律。上述结果对新疆野苹果的保护工作有重要的参考意义。

Malus sieversii is the ancestor of apple,and it is an important genetic resource for breeding and usually used as rootstock.In recent years,the distribution area of M.sieversii has plummeted,the protection of M.sieversii is becoming more and more important.Here,we adopted high-throughput sequencing technology to detect viruses from 26 samples collected from the wild fruit forest in Tianshan Mountain,Ili,Xinjiang and found the infections of apple chlorotic leaf spot virus(ACLSV)and apple stem pitting virus(ASPV).Among 127 samples,the infection of ACLSV,ASPV and apple stem grooving virus(ASGV)were detected using RT-PCR,with the detection rates of 22%,19.7%,and 11%,respectively.Using RACE PCR,the complete genome of ASGV from M.sieversii was amplified,and tentatively named ASGV-XJ.The complete genomic sequence of this virus was 6506 bp excluding the 3′poly(A)tail,encoding two overlapping open reading frames(ORFs).ORF1(position 47-6364 nt)encodes a 241 kD polyprotein,including domains of a methyltransferase,a papain-like-protease,a nucleotide triphosphate-binding helicase,a RNA-dependent RNA polymerase,and a coat protein.ORF2(4798-5760 nt)encodes a movement protein.Phylogenetic analysis indicated that no geographic or host association among the isolates.These results remind us that virus infection cannot be neglected during the protection of M.sieversii.