Abstract:

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 HiSeq^TM 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 277 048 525 clean reads, including 82.86 G nucleotides, were obtained. After assembly, 44 878 unigenes were obtained, with an average length of 1 082.76 bp and an N50 of 1 976 bp. A total of 14 190 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.

Key words: Leptocybe invasa, transcriptome sequencing, EST-SSR, cryptic species identification