Pushing the limits of whole genome amplification: successful sequencing of RADseq library from a single microhymenopteran (Chalcidoidea, Trichogramma)

Abstract

A major obstacle to high-throughput genotyping of microhymenoptera is their small size. As species are difficult to discriminate, and because complexes may exist, the sequencing of a pool of specimens is hazardous. Thus, one should be able to sequence pangenomic markers (e.g., RADtags) from a single specimen. To date, whole genome amplification (WGA) prior to library construction is still a necessity as at most 10 ng of DNA can be obtained from single specimens (sometimes less). However, this amount of DNA is not compatible with manufacturer's requirements for commercial kits. Here we test the accuracy of the GenomiPhi kit V2 on Trichogramma wasps by comparing RAD libraries obtained from the WGA of single specimens (F0 and F1 generation, about1 ng input DNA for the WGA (0.17-2.9 ng)) and a biological amplification of genomic material (the pool of the progeny of the F1 generation). Globally, we found that 99% of the examined loci (up to 48,189 for one of the crosses, 109 bp each) were compatible with the mode of reproduction of the studied model (haplodiploidy) and Mendelian inheritance of alleles. The remaining 1% (0.01% of the analysed nucleotides) could represent WGA bias or other experimental/analytical bias. This study shows that the multiple displacement amplification method on which the GenomiPhi kit relies, could also be of great help for the high-throughput genotyping of microhymenoptera used for biological control, or other organisms from which only a very small amount of DNA can be extracted, such as human disease vectors (e.g., sandflies, fleas, ticks etc.).

Keywords: GenomiPhi; High-throughput genotyping; Microarthropods; RAD; Small amount of DNA; WGA.

Figure 1. Experimental setup.

In Trichogramma, females develop from fertilized eggs and are diploid, while males develop from unfertilized eggs and are haploid. In this figure, the coloured bars close to the female and male symbols represent alleles. The breeding experiment was as follows: (1) A female/male pair (F0 generation) was left free to mate in a glass tube (one pair per tube). Sterilized eggs of E. kuehniella (Pyralidae) were provided for oviposition. (2) Only females of the F1 generation were kept, males were discarded. Virgin F1 females were isolated into glass tubes (one female per tube). Again, sterilized eggs of E. kuehniella (Pyralidae) were provided for oviposition. (3) All F2 males were kept. This breeding experiment was replicated ten times. For clarity and to provide allele frequency predicted by Mendel’s laws of inheritance, only two F1 females are represented on the figure. Similarly, only two F2 males are represented for each F1 female. For each replicate, a WGA was performed prior to RAD library construction on the F0 female, the F0 male and one F1 female, while all F2 males were pooled prior to DNA extraction and the resulting DNA was directly used as input for RAD library construction without WGA. Thus, the F2 generation was used as a negative control. Photo T. brassicae male ©J-Y Rasplus.