Large-scale analysis of Macaca fascicularis transcripts and inference of genetic divergence between M. fascicularis and M. mulatta

Abstract

Background: Cynomolgus macaques (Macaca fascicularis) are widely used as experimental animals in biomedical research and are closely related to other laboratory macaques, such as rhesus macaques (M. mulatta). We isolated 85,721 clones and determined 9407 full-insert sequences from cynomolgus monkey brain, testis, and liver. These sequences were annotated based on homology to human genes and stored in a database, QFbase http://genebank.nibio.go.jp/qfbase/.

Results: We found that 1024 transcripts did not represent any public human cDNA sequence and examined their expression using M. fascicularis oligonucleotide microarrays. Significant expression was detected for 544 (51%) of the unidentified transcripts. Moreover, we identified 226 genes containing exon alterations in the untranslated regions of the macaque transcripts, despite the highly conserved structure of the coding regions. Considering the polymorphism in the common ancestor of cynomolgus and rhesus macaques and the rate of PCR errors, the divergence time between the two species was estimated to be around 0.9 million years ago.

Conclusion: Transcript data from Old World monkeys provide a means not only to determine the evolutionary difference between human and non-human primates but also to unveil hidden transcripts in the human genome. Increasing the genomic resources and information of macaque monkeys will greatly contribute to the development of evolutionary biology and biomedical sciences.

Figure 1

Classification of the 1245 Non-RefSeq transcripts. Transcripts shorter than 300 bp after masking the repetitive sequences were categorized as junk sequences. The remaining sequences were BLAST-searched against all public human cDNA sequences for the forward strand. Homologous sequences to the unannotated human cDNAs were classified as orphan transcripts for the forward strand and anti-transcript for the reverse strand. The remaining 947 clones were mapped on the human genome sequence and arranged according to the annotation from the UCSC genome browser (hg18). The transcripts that overlapped with the genic regions including UTR were classified as intronic transcripts, and the transcripts that were mapped more than 5 kb away from the genic region were classified as intergenic transcripts.

Figure 2

The proportion of the expressed transcripts in the RefSeq homologs (control) and unidentified transcripts. Cerebrum, cerebellum, liver, and testis of a male macaque were used for the microarray experiments with duplicated hybridizations. The transcripts were classified into no expression (blue), expressed in 1–3 tissues (grey), or expressed in all tissues (red).

Figure 3

Sequence conservation of the brain-expressed and testis-expressed transcripts between humans and macaques. For the RefSeq homologs (control), the non-synonymous (K_a) and synonymous (K_s) substitution rates were estimated using the Li-Pamilo-Bianchi method [48]. The substitution rates in the intergenic and intronic transcripts were estimated using Kimura's two parameter methods [55]. The heights of the boxes represent the lower and upper quartile points, and the whiskers show the minimum and maximum points.

Figure 4

Distribution of transcript expression levels of the RefSeq homologs (blue) and the intergenic transcripts (red). Only the transcripts that were determined as significantly expressed on the microarray are presented in the figure. Log-transformed signal intensity in the tissue with the highest expression was shown. The intergenic transcripts showed significantly lower expression levels than the RefSeq homologs.

Figure 5

RT-PCR gel images for the expression of the intergenic transcripts in the human (H) and the macaque (Q) brain. Transcript names indicate whether the expression was detected by the microarray experiments (red) or not (blue). Expected PCR products are marked by the white arrows.

Figure 6

Pattern of the unidentified exons. The closed boxes represent exons in the genomes. Unidentified exons in macaques are presented as blue boxes. Intergenic regions and introns are depicted by thick and thin horizontal lines, respectively. (A) extended exons. (B) novel exons. These exons were further classified into internal (right panel) and external (left panel) exons. The number of genes in each category is shown on the left of each schema. The number of unidentified exons that have not been found even in the EST sequences is shown in parentheses.

Figure 7

Genealogical relationship (phylogeny of genes) among the humans (H), cynomolgus macaque (C), and rhesus macaque (R). The common ancestor of the two macaques is indicated by the letter O. The time of speciation between the two macaques is shown by the dashed line. Note that the tree is unrooted.