Wang et al. BMC Genomics 2012, 13:529 http://www.biomedcentral.com/1471-2164/13/529 Analysis of SNP To further understand the mechanism of divergence, we analyzed the potential SNP sites in CDS of the ortholo- gous gene pairs between Asia II 3 and MEAM1. For Asia II 3, a total of 138 SNPs were identified within the 1433.63 kb aligned regions, about 1 every ten thousands bp. The complete list of SNPs with annotation can be found in Additional file 10. Of the 138 SNPs, 96 (69.6%) were synonymous and 42 (30.4%) were non-synonym- ous. This percentage of SNPs is much lower than those obtained in previous analyses in other insects [41,42]. The possible reason is that our B. tabaci populations were established from a pair of B. tabaci and the sam- ples were collected within five generations. For MEAM1, a slightly higher number of SNPs (248, about 1.7 every ten thousands bp) were found in the orthologous gene pairs with 196 synonymous and 52 non-synonymous (Additional file 10). Compared with the average diver- gence between Asia II 3 and MEAM1 in CDS (1.83%), the percentage of SNPs is more than 100 times lower. Next, the potential SNP sites in the CDS of orthologous gene pairs between Asia II 3 and MED were analyzed. Our results showed that a total of 56 SNPs in Asia II 3 and 627 SNPs in MED were identified within the 1,071 kb aligned regions (Additional file 11). The large vari- ation between the numbers of SNPs in Asia II 3 and MED is probably due to the difference in the sequencing among of Asia II 3 (1G) and MED (3G) (Additional file 1). Some of the SNPs in Asia II 3 might have been fil- tered out because only SNP sites with the minimum read depth of 10 reads were selected. Thses results are con- sistent with previous findings, in which overall number of SNPs decreases at lower coverage levels [43]. While the SNPs we have identified here are suitable for future research, more rigorous statistical tests are required to confirm the current results as well as to detect specific codons undergoing adaptive changes. In addition, fur- ther studies of the SNPs on population samples are war- ranted as our data were generated from inbred lab colonies. Conclusions In summary, this study dramatically increases the num- ber of genes from the native Asia II 3 B. tabaci species. Together with the previously available MEAM1 and MED transcriptomes, this study is the first globally com- parative analyses of the genetic differences between the invasive and indigenous B. tabaci species. Based on se- quence homology, a group of 3,023 protein families con- served among the Asia II 3, MEAM1 and MED species were identified. These protein families might be respon- sible for core cellular and physiological functions of the B. tabaci complex. Sequence comparisons of all ortholo- gous gene pairs revealed that the average genetic Page 9 of 13 divergences between Asia II 3 and invasive MEAM1 are nearly twice of that between MEAM1 and MED, in ac- cordance with previous genetic studies. The divergent genes identified in this study will be an invaluable re- source to reveal the possible mechanisms of B. tabaci in- vasion, displacement and speciation. Methods Insect rearing Stock culture of the Asia II 3 (mitochondrial cytochrome oxidase I gene GenBank accession no: AJ867556) was maintained on cotton, Gossypium hirsutum (Malvaceae) cv. Zhe-Mian 1793 in a climate chamber (see conditions below). The purity of the culture was monitored using the random amplified polymorphic DNA-PCR technique with the primer H16 (5’-TCTCAGCTGG-3’) [44]. For sample preparation, a pair of virgin adults of B. tabaci Asia II 3 were released onto a cotton plant to oviposit and develop for five generations at 27 ± 1°C, a photo- period of 14 h light:10 h darkness and 70 ± 10% relative humidity [45]. The same protocols were used to raise the MEAM1 and MED whiteflies for sample collection and subsequent transcriptome data generation. Sample preparation and RNA isolation In order to get an overall picture of the Asia II 3 whitefly transcriptome, we collected the samples from four differ- ent developing stages: 1) egg & nymph (the eggs are ex- tremely small, therefore a mixture of eggs and first to third instar nymphs were collected as one sample); 2) pupa; 3) female adult and 4) male adult. To ensure that the whitefly adults are in the same developmental stage, only newly emerged adults were collected. Previously, samples from MEAM1 and MED have been collected using the same strategy [29,30]. Total RNA was isolated from the four samples using SV total RNA isolation sys- tem (Promega) according to the manufacturer’s protocol [46]. RNA integrity was confirmed using the 2100 Bioa- nalyzer (Agilent Technologies) with a minimum RNA integrated number value of 8. Then, equal amount of RNA from egg & nymph, pupa, female adult and male adult were mixed, and mRNA was purified from the mixture using oligo (dT) magnetic beads. Library preparation and Illumina sequencing For transcriptome sequencing, a 200 bp cDNA library was prepared using Illumina’s kit as previously described [29]. The library was not normalized because we intend to use the resulting sequence reads to analyze the level of gene expression. The cDNA library was sequenced for both ends on the GAII Illumina sequencing platform (a single lane) at The Beijing Genome Institute (Shenzhen, China). The total sequencing amount was 1G. The raw reads were filtered by removing adaptor sequences,