入侵性致瘿害虫桉树枝瘿姬小蜂(膜翅目:姬小蜂科)EST-SSR开发及隐种鉴定

EST-SSR Development and Cryptic Species Identification of the Invasive Gall-Causing Pest Leptocybe invasa(Hymenopetra:Eulophidae)

【目的】对入侵性致瘿害虫桉树枝瘿姬小蜂(膜翅目:姬小蜂科)进行EST-SSR引物开发和隐种鉴定,为桉树枝瘿姬小蜂种群遗传学研究及其防治提供理论依据。【方法】采用Illumina HiSeqTM 2000测序平台对3个地理种群的桉树枝瘿姬小蜂雌性成虫进行转录组测序,利用MISA和Primer Premier 3软件进行EST-SSR标记的搜索、挖掘和引物设计。选取400对EST-SSR引物,结合国外报道的14对多态性G-SSR引物,应用1%琼脂糖凝胶电泳检测扩增效率,并用8%变性聚丙烯酰胺凝胶电泳验证引物多态性。基于桉树枝瘿姬小蜂COI基因序列数据对我国14个地理种群320个桉树枝瘿姬小蜂雌性成虫样本进行隐种鉴定。【结果】1)共获得277048525条clean reads,包括82.86 G个核苷酸,组装后得到44878个unigene,平均长度1082.76 bp,N50为1976 bp;利用MISA软件搜索到EST-SSR位点14190个,其中主要重复类型是单核苷酸重复(占总EST-SSR的69.63%),其次是二核苷酸重复和三核苷酸重复(分别占总EST-SSR的16.17%、13.51%)。2)400对EST-SSR引物中有205对引物可有效扩增出目的片段,扩增效率51.25%,国外报道的14对G-SSR引物均能扩增出目的片段;3)用8%变性聚丙烯酰胺凝胶电泳验证SSR引物多态性,最终筛选出10对多态性良好的EST-SSR引物,国外报道的14对G-SSR引物中仅有LiSS2、LiSS5、LiSS13在我国桉树枝瘿姬小蜂样本中存在多态性;4)从我国14个桉树枝瘿姬小蜂地理种群中共获得320条605 bp的COI基因序列,基于桉树枝瘿姬小蜂COI基因序列构建的NJ系统发育树表明,我国仅存在A、B 2种类型的桉树枝瘿姬小蜂隐种,样本比例约为1∶2。【结论】筛选出10对适合我国桉树枝瘿姬小蜂种群遗传学研究的EST-SSR引物。我国存在A、B 2种类型的桉树枝瘿姬小蜂隐种,其中隐种A首次在我国发现,桉树枝瘿姬小蜂的隐种鉴定可为该害虫在生物防治中采用正确的生物型提供依据。

【Objective】Leptocybe invasa(Hymenopetra:Eulophidae)is a global invasive pest that causes serious damage to eucalyptus trees by forming galls on the twigs and leaves.The purpose of this paper is to provide a theoretical basis for the genetic study and control it by the development of EST-SSR primers and cryptic species identification.【Method】The Illumina HiSeqTM 2000 sequencing platform was used to perform transcriptome sequencing of female adults of L.invasa from 3 geographical populations.The search,tapping and primer design of EST-SSR markers were carried out by using MISA and Primer Premier 3 software,and 400 EST-SSR primer pairs were selected.Combined with 14 pairs of polymorphic G-SSR primers reported abroad,The amplification efficiency was detected by 1%agarose gel electrophoresis,and the polymorphism of the primers was verified by 8%denatured polyacrylamide gel electrophoresis.The cryptic species identification of 320 female adults from 14 geographic populations in China was conducted based on the COI gene sequence data.【Result】1)A total of 277048525 clean reads,including 82.86 G nucleotides,were obtained.After assembly,44878 unigenes were obtained,with an average length of 1082.76 bp and an N50 of 1976 bp.A total of 14190 EST-SSR loci were searched using MISA software,of which the major repeat type was mononucleotide repeats(69.63%of the total EST-SSRs),followed by dinucleotide and trinucleotide repeats(16.17%,13.51%of the total EST-SSRs,respectively).2)A total of 205 primer pairs out of 400 EST-SSR primers were used to effectively amplify the target fragment,and the amplification efficiency was 51.25%,whereas abroad 14 primer pairs of G-SSR were all able to amplify the target fragment.3)Polymorphism of SSR primers was verified by 8%denatured polyacrylamide gel electrophoresis,and 10 pairs of EST-SSR primers with good polymorphism were finally screened.Among the 14 pairs of G-SSR primers in foreign literature,only LiSS2,LiSS5,and LiSS13 showed polymorphism in L.invasa samples in China.4)A total of 320 COI gene sequences of 605 bp were obtained from 14 geographic populations of L.invasa in China.The NJ phylogenetic tree constructed based on the COI gene sequence of L.invasa showed that there were only cryptic species A and cryptic species B in China,with a sample ratio of about 1∶2.【Conclusion】In this study,10 pairs of EST-SSR primers suitable for the population genetics study of L.invasa in China have been developed.There are two types of cryptic species of L.invasa cryptic species A and cryptic species B,in China,of which the cryptic species A is found in China for the first time.The identification of cryptic species of L.invasa can provide a basis for the use of correct biological types in biological control of it.