Large-scale analysis of Macaca fascicularis transcripts and inference of genetic divergence between M. fascicularis and M. mulatta
© Osada et al; licensee BioMed Central Ltd. 2008
Received: 27 September 2007
Accepted: 24 February 2008
Published: 24 February 2008
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/.
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.
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.
Genomic resources and information about primates are valuable for evolutionary and biomedical studies to determine how and why phenotypes specific to humans, as well as human diseases, have been formed. Moreover, they are important for extrapolating the results of laboratory experiments to medical research because the physiology of primates is more similar to that of humans as compared with other common experimental animals such as rodents. The cynomolgus macaque (Macaca fascicularis), also known as the long-tailed or crab-eating macaque, is an Old World monkey living in Southeast Asia. It is bred in laboratories worldwide and is one of the most popular primates used for laboratory animal studies, such as those on infectious diseases, immunology, pharmacology, tissue engineering, gene therapy, senescence, and learning . Cynomolgus macaques, rhesus macaques (M. mulatta), and Japanese macaques (M. fuscata) are widely used for experimental studies and are closely related to each other [2–4]. The US government funded genome sequencing of the rhesus macaque because it is the most common laboratory animal bred in the US, and in 2007, the draft sequence of the rhesus macaque was published .
Since cynomolgus and rhesus monkeys are very closely related at the genetic level, we aim to determine the extent to which the rhesus macaque genome sequence can be used as a reference for biomedical studies involving cynomolgus macaques. At the chromosomal level, a previous study suggested that a pericentric chromosome inversion occurred in the cynomolgus lineage after splitting from rhesus macaques . At the nucleotide sequence level, the genetic divergence between cynomolgus and rhesus monkeys has been measured using mitochondrial DNA sequences [2, 3] or a limited number of loci on the chromosomes [4, 7]. Thus, the divergence of a sufficient number of loci between cynomolgus and rhesus macaques would assist in determining the degree of genetic divergence between them. In addition, recent studies have shown that there is a considerable amount of genetic diversity within the species themselves [5–10], which also hampers the measurement of the genetic divergence. Because the divergence between the two macaques is very recent (much later than the divergence between humans and chimpanzees), we must consider the segregation of polymorphisms in the common ancestral population to estimate the correct species divergence time [11, 12]. By analyzing the number of loci in the two species, we can determine the history of divergence between them, including the ancestral population size, divergence time between species, and possible gene flow [13, 14].
We have constructed full-length-enriched cDNA libraries from cynomolgus monkey brain, testis, and liver using the oligo-capping method. Many comparative genomics projects have focused on sequencing of the genome or expressed sequenced tags (ESTs), and full-length cDNA sequences are uniquely informative resources for accurately predicting the full structure of transcripts in the genome . Furthermore, because cynomolgus and rhesus macaques are very closely related, transcriptome data from cynomolgus macaques is useful for annotating the genome sequence of other macaques whose transcriptome data is less than 1% of that from humans and whose full-length cDNA data is scarce.
Along with the cynomolgus macaque cDNA sequencing project, we have published a part of our results, such as novel gene findings [16–19], search for fast-evolving genes , molecular evolution of 5'-untranslated regions (UTRs) , and evolution of brain-expressed genes . In this study, we summarize the final sequencing project and present novel findings with an expanded dataset. In total, 85,721 ESTs and 9407 full-length sequences were determined, annotated, and stored in an in-house database and the public databases (DDBJ/EMBL/GenBank). Our study focused on the divergence between the cynomolgus and rhesus macaque genes. We did not intensively analyze the divergence between humans and cynomolgus monkeys, because a study on rhesus genome has investigated this thoroughly ; it also identified and discussed positively selected genes or extensively duplicated genomic regions during the evolution of Catarrhine primates.
Summary of cDNA sequences
Summary of cDNA clones
# of isolated clones
# of full-sequenced clones
Brain: Parietal Lobe (QnpA)
Brain: Frontal Lobe (QflA)
Brain: Temporal Lobe (QtrA)
Brain: Occipital Lobe (QorA)
Brain Stem (QbsA, B)
Brain: Medulla Oblongata (QmoA)
Brain: Cerebellar Cortex (QccE)
In parallel to EST sequencing, we determined about 9500 full-insert sequences of the cDNA clones. About 2500 clones whose 5'-EST sequences were not homologous to the public cDNA sequences and 7000 clones whose 5'-EST sequences were homologous to the human RefSeq sequences were chosen [16–21]. Out of the 9407 full-insert sequences, 7407 sequences were homologous to 5384 types of human genes (Table 1). The averaged length of the full-insert sequences was 1864 bp, excluding the length of the poly(A) tail. The macaque sequences were annotated for gene function and homologous locus in the human genome using information from the Entrez Gene  and Gene Ontology (GO) databases .
All cDNA sequences and annotations were deposited in the public databases and stored in a simple in-house database, QFbase . On the QFbase website, users can search the macaque clones by keywords and BLAST searches. For each human gene, the distribution of the macaque homologs is represented graphically and users can easily retrieve information of the objective macaque cDNA clones. The entries are further linked to the gene annotation in the outside databases, GenBank , Ensembl , OMIM , and H-InvDB . The cDNA sequences were mapped on the human and rhesus genome sequences using the UCSC genome browser . Moreover, 4665 human-macaque orthologous alignments are provided in the QFbase. For each alignment, the non-synonymous substitution rate (K a ) and the synonymous substitution rate (K s ) between the human and macaque cDNA sequences were estimated. Non-synonymous substitutions are nucleotide changes that replace amino acids between species whereas synonymous substitutions cause no amino acid changes. The relative pace of protein evolution was thus determined using K a /K s , assuming that the K s value reflects the neutral mutation rate . Using the database, users can sort the alignments according to the K a and K s values. For example, users can determine the K a and K s values of a particular gene or view the list of the 100 most rapidly evolved genes between humans and cynomolgus monkeys. The cDNA clones are distributed through the Human Science Research Resource Bank in Japan (Tokyo, Japan). Further information is available at the QFbase website.
Analysis of unidentified transcripts
After removing the junk sequences and orphan transcripts, the remaining 1024 transcripts were referred as the unidentified transcripts although 40% (406/1024) of the transcripts showed homology to human ESTs (BLAST: E = 1e-60), because no full cDNA sequence of humans has been registered in the public databases. One of the advantages of full-length cDNAs is that we can determine the splicing pattern and reading direction of the transcripts in the genome. We categorized the unidentified transcripts as anti-transcript, intronic spliced transcript, intronic single-exon transcript, intergenic spliced transcript, or intergenic single-exon transcript. Among the intergenic transcripts, 82 were located within 5 kb of the genic regions with the same direction as the genes. Of these, 6 were mapped on the upstream regions and 76 were mapped on the downstream regions of the known genes. The result showed they may be hidden extensions of the known transcripts, using alternative promoters and/or poly(A) signals in the human genome. These sequences were filtered from the intergenic transcripts and classified as 'flanking' to genic regions. The largest group was the intronic single-exon transcripts. Although they might be acquired from premature mRNA molecules in the cell nucleus, recent studies have revealed the potential abundance of short intronic transcripts in the human genome . Among these classes, anti-transcripts and intergenic spliced transcripts are the most biologically relevant classes, which are unlikely to be derived from contamination by premature mRNAs.
Number of expressed transcripts in the unknown macaque transcripts
Hidden transcript structures in the human genome
Because the human transcriptome data is more complex than previously thought, as revealed by genome tiling DNA microarrays [34–36], these unrepresented exons may be expressed at a very low level in human tissues. Moreover, these exons have not been found in the conventional cDNA exploration methods. However, previous studies have suggested a frequent evolutionary turnover of exon sequences . The evolutionary alteration of external exons in the 5'-UTR may be caused by the alternative usage of promoter sequences . The evolutionarily altered exons in the 3'-UTR may be caused by the alternative usage of poly(A) adenylation signals . All the unidentified exons are provided in Additional file 2.
Comparison of the human, cynomolgus, and rhesus genes
Number of genes under positive selection out of 1499 non-duplicated genes determined using the branch-site test of positive selection
P ≤ 0.05
P ≤ 0.01
Between the macaques (C-O + R-O)
All lineages (H-O + C-O + R-O)
Genetic divergence between cynomolgus and rhesus macaques
Divergence among the human, cynomolgus, and rhesus genes
Model without ancestral polymorphisms (Raw data)
K a (± S.E.)
K s (± S.E.)
1.06 × 10-2 (3.20 × 10-4)
6.82 × 10-2 (1.07 × 10-3)
1.02 × 10-3 (4.79 × 10-5)
3.04 × 10-3 (1.20 × 10-4)
4.98 × 10-4 (3.36 × 10-5)
2.50 × 10-3 (1.15 × 10-4)
Model with ancestral polymorphisms
4N e ub (± S.E.)
2.13 × 10-3 (2.24 × 10-4)
3.27 × 10-3 (2.52 × 10-4)
PCR error corrected
1.81 × 10-3 (2.12 × 10-4)
3.11 × 10-3 (2.40 × 10-4)
In summary, the sequencing project of cynomolgus monkey cDNAs yielded 85,721 ESTs and 9407 full-length sequences. Since our project mainly studied the brain and testis, the dataset is deficient in other tissue-specific genes, e.g., the genes related to the immune system that many medical researchers would want to explore . The construction of cDNA libraries from other tissues and EST sequencing is still ongoing to complement the transcriptome of cynomolgus monkeys. The latest sequencing status can be confirmed from the website. Because of the close relationship between the cynomolgus and rhesus macaques, cDNA resources of cynomolgus macaques not only are useful for research using cynomolgus macaques but also complement the relative paucity of the transcriptome data from rhesus macaques. Using macaque tissues to scan the primate transcriptome is advantageous because RNA molecules are unstable and are instantly degraded in the tissues during sampling. This causes serious problems for RNA sampling from human tissues, especially in the brain, where fresh samples are rarely obtainable. Therefore, we hope to uncover rare transcripts that would be hidden in the human transcriptome data. In this study, we identified 1024 macaque cDNAs that were not represented in the public human cDNA sequences. Although 51% of the cDNA did not show a positive signal on the microarrays, the following RT-PCR experiments recovered the expression in half (3/6) of the transcripts. The results indicate that these unidentified transcripts were expressed at a low level in the tissues even though the microarray could not detect the expression.
The M. fascicularis oligonucleotide microarrays contain probes that matched 8316 known genes and 1024 unidentified transcripts. We determined the number of probe sets for the known genes that overlapped among the commercially available microarray (Affymetrix GeneChip) and previously published microarray of rhesus macaque by Wallace et al., which contains the largest number of probe sets among the published microarrays . Of our 8316 probes for the known genes, 1728 (21%) were not represented in the commercial microarray and 1091 (13%) were not found in the published microarray. Combining the three microarrays, 417 probes for the known genes were represented only in the M. fascicularis microarrays [see Additional file 5]. In our preliminary study of the polymorphisms within cynomolgus macaques, we found that the level of polymorphisms in cynomolgus macaques was greater than that in rhesus macaques and slightly smaller than the level of divergence between rhesus and cynomolgus macaques (Osada et al., unpublished data). Therefore, even if we should be careful about sequence mismatches within and between species, the information from both macaque transcripts and the rhesus genome can be combined to build more versatile and comprehensive DNA microarrays that can be used for biomedical surveys using laboratory macaques.
Suppose that we identify positively selected genes in the human lineage after the spilt from chimpanzees. Such genes are useful for understanding the human-specific physiology only when those genes have not been under positive selection in other primate lineages. We identified 37 genes under positive selection between the two macaques at 5% significance level. None of these genes were shared with 387 genes under positive selection in the human or chimpanzee lineages previously determined from the whole genome scan , providing support that the method has correctly identified positively selected genes in the specific lineages.
For estimating of the divergence time between cynomolgus and rhesus macaques, we assumed that there is no gene flow between the ancestral species throughout their speciation and divergence time (i.e., allopatric model). However, considering the ancestral polymorphisms and the PCR error rate, we estimated the divergence time to be around 0.9 Mya, which is less than the estimation of the age of MRCA of rhesus macaques . Indeed, more than half of the genetic divergence between the two macaques was derived from ancestral polymorphisms. If continuous gene flow is present during speciation, the variance component would be inflated and we would tend to overestimate the amount of ancestral polymorphisms .
In this analysis, we used the rhesus macaque genome sequence to represent rhesus macaques. We should note that the rhesus macaque used for genome sequencing was an Indian rhesus macaque; these macaques have genetically differentiated from Chinese rhesus macaques . In addition, our samples of cynomolgus macaques were obtained from different geographic subpopulations. Previous studies using mitochondrial DNA sequences  and our preliminary analysis using nuclear DNA sequences (Osada et al., unpublished data) showed that there is a substantial genetic divergence between cynomolgus monkeys of Sundaland (Indonesia and Indochina) and Philippine populations. Therefore, our phylogenetic inference using two sampled sequences has a technical limitation and may be accurate only if there are no complex population structures among the ancestral cynomolgus and rhesus macaque populations. Elucidating the polymorphisms and divergence among macaque species would provide further insight into the evolutionary history of macaques and benefit biomedical research using macaque monkeys.
In Table 4, without correcting the PCR error rate, both the non-synonymous and synonymous divergences are greater in the cynomolgus lineage. This may be due to shorter generation time and smaller population size of cynomolgus monkeys. However, a more reasonable explanation is that the cDNA sequences of cynomolgus monkeys might incorporate the errors resulting from PCR amplification during the construction of the oligo-capped cDNA libraries. The synonymous substitution rate in the cynomolgus lineage is about 0.0005 points higher than that in the rhesus lineage, and the non-synonymous substitution rate differs in about 0.0004 points. Assuming that the selective constraint and generation time of the two macaque lineages are the same, excess divergence of 0.04%-0.05% in the cynomolgus lineage may be an artifact introduced by PCR amplification, which is fairly close to the estimation from the experiment by Suzuki and Sugano . If we reflect the substitution rate in the rhesus lineage to that in the cynomolgus lineage for correcting the errors, the total divergence of the two macaques will be reduced to about 90% (Table 4).
Transcript data from Old World monkeys provide us with means to determine not only the evolutionary difference between human and non-human primates but also the hidden transcripts in the human genome. Actual cDNA clones of macaques are also indispensable resources for genetic engineering studies. It is considered that the species divergence between rhesus and cynomolgus macaques would be much later than the previous estimates, and the speciation process between them might have been complex. To use laboratory macaques more efficiently, we need to be more aware of the genetic difference within and among macaque monkeys. Increasing the genomic resources and information of macaque monkeys will greatly contribute to the development of evolutionary biology and biomedical sciences.
Cynomolgus monkey samples
Samples from two cynomolgus monkeys, a 16-year-old female (Philippine origin) and a 15-year-old male (Cambodian-Thai hybrid), were used for the cDNA libraries, except for the liver cDNA library (Qlv). The liver samples were collected from three adult cynomolgus monkeys of unknown origin. The monkeys were cared for and handled according to the guidelines established by the Institutional Animal Care and Use Committee of the National Institute of Infectious Diseases (NIID) of Japan and the standard operating procedures for monkeys at the Tsukuba Primate Center, NIID (present National Institute of Biomedical Innovation), Tsukuba, Ibaraki, Japan. Tissues were excised in accordance with all the guidelines in the Laboratory Biosafety Manual, World Health Organization, at the P3 facility for monkeys of the Tsukuba Primate Center. Immediately after collection, the tissues were frozen in liquid nitrogen and used for RNA extraction. Oligo-capped cDNA libraries were constructed according to the method described previously . The prefix in each clone name represents the location of the source of the tissue: Qnp (brain, parietal lobe), Qfl (brain, frontal lobe), Qtr (brain, temporal lobe), Qor (brain, occipital lobe), Qbs (brain stem), Qmo (medulla oblongata), Qcc (cerebellar cortex), Qlv (liver), and Qts (testis).
Sequencing of cDNA clones
The cDNA clones were sequenced with ABI 3700 and 3730 automated sequencers. The EST sequences were trimmed to avoid the vector sequence of pME18-FL3 [DDBJ/EMBL/GenBank: AB009864]. Entire sequences of the clones were determined by the primer walking method. The repeat sequences at the 5'- and 3'-ends were masked using the Repbase Update database  before BLAST search. The BLAST search was performed with an e-60 cut-off value against non-redundant human RefSeq data. The non-redundant data was based on the annotation in the Ensembl Gene database. The longest transcript in the locus was selected as the representative cDNA [24, 50]. The macaque cDNA sequences were deposited in the public DNA databases [DDBJ/EMBL/GenBank: CJ430287–CJ493524; BB873801–BB894695; AB303966–AB303967].
Classification of unidentified transcripts
Classification of the Non-RefSeq transcripts was performed as shown in Figure 1. 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 (downloaded on Aug 3, 2007) for the forward strand. Homologous sequences to the 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 (build 36.1) by BLAST algorithm 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.
Affymetrix GeneChip was designed using the available cDNA sequences of M. fascicularis. The chip loads 10,307 probe sets. RNA samples from the cerebrum, cerebellum, liver, and testis of a 3-year-old male cynomolgus monkey were extracted using TRIZOL (Invitrogen) and hybridized to the GeneChip with duplication in a single experiment. The M. fascicularis GeneChip contains at most 11 perfect-match probes (25-mers complete matches to the cDNA sequences) and 11 mismatch probes (containing one mismatched oligo) for each probe set, similar to other GeneChip formats. Normalization, signal detection, and signal intensity calculation of the microarrays were performed using Affymetrix MAS5.0 software. Transcripts were considered as expressed when the probe set of both the duplicates agreed for the significant expression (P ≤ 0.05) . The raw array data were deposited in Gene Expression Omnibus [GEO: GSM201873–201880]. The array design and the sequences of oligonucleotide probes were deposited in the public database [GEO: GPL5396].
Templates of the human brain RNA were purchased from Clontech. The macaque brain RNA was obtained from a 21-year-old male cynomolgus monkey. One microgram of total mRNA was amplified using the PrimeSTAR® RT-PCR Kit (TakaraBio). The temperature and time schedules were 30 cycles at 94°C for 20 s, 60°C for 30 s, and 72°C for 1 min. All primer sequences are presented in Additional file 6.
Human-cynomolgus cDNA sequence alignment
Human-macaque orthologous gene pairs were assigned by the reciprocal best BLAST hit with an e-60 cut-off value. We aligned only that part of the coding sequences (CDS) that was homologous to a BLAST search, because representative human and macaque cDNAs do not necessarily have the same splicing isoforms. The sequences of human and macaque cDNAs were aligned using CLUSTAL W , and an unaligned macaque nucleotide was marked by the letter X in the database. Alignments shorter than 100 bp (≤ 33 codons) were filtered for further analysis. In the database, the positions including deletion in the human sequence (or insertion in the macaque sequence) were dropped for estimating the substitution rates. The non-synonymous substitution rate per non-synonymous site (K a ) and the synonymous substitution rate per synonymous site (K s ) were estimated using the Li-Pamilo-Bianchi method [52, 53]. K a /K s ratios were set to 100 in the database when the K s value was zero.
Human-rhesus-cynomolgus cDNA sequence alignment
The predicted cDNA sequences of rhesus macaques were downloaded from Ensembl (MMUL1.0) and aligned with the human RefSeq sequences. Orthology between the rhesus and cynomolgus genes was confirmed again using the cynomolgus-rhesus reciprocal BLAST hit, and human-rhesus-cynomolgus cDNA alignments were compiled. Alignments containing any frameshifting indels and those shorter than 100 bp (≤ 33 codons) were filtered, which resulted in 2655 alignments (dataset I). The rhesus cDNA sequences were then mapped on the draft genome sequence of the rhesus macaque (rheMac2). The rhesus macaque genes showing > 80% homology to more than one locus on the rhesus genome were removed from the alignments, which yielded 1499 human-rhesus-cynomolgus cDNA alignments (dataset II). To estimate the divergence among the three species, K a (d n ) and K s (d s ) were estimated using the maximum likelihood method implemented in the PAML program package . We estimated the transition/transversion ratios in 4-fold degenerated sites using the concatenated cynomolgus and rhesus alignments in advance, and fixed the value to the observed value. The test of positive selection was conducted using the branch-site test of positive selection described by Zhang et al. , applying the critical values of 2.71 and 5.41 at 5% and 1% significance level without a Bonferroni correction, respectively.
Estimation of the divergence time between cynomolgus and rhesus macaques
Maximum likelihood estimation (MLE) of the divergence time and ancestral population size was performed using the method of Takahata and Satta [11, 14]. MLE was determined using the Newton-Raphson algorithm with many possible initial values. The standard error was determined from the numerically evaluated Fisher information matrix. In order to correct a PCR error rate, we estimated the PCR error rate to be 5.40 × 10-4, which was derived from the difference in the synonymous substitution rates of the cynomolgus and rhesus lineages. We assumed that the generation time and the effect of selection on the synonymous sites of the two macaques were the same, and that the erroneous nucleotide incorporated by PCR did not skew. Therefore, when a synonymous substitution in the cynomolgus lineage was found, it was considered that the substitution is because of the PCR error with a probability of 0.178 (5.40 × 10-4/3.04 × 10-3). We randomly corrected the number of substitutions in the raw data, generated pseudo data for 1000 times, and estimated the evolutionary parameter for each time.
expressed sequence tag
maximum likelihood estimation
open reading frame
most recent common ancestor
This study was supported by a Health Science Research Grant from the Ministry of Health, Labor and Welfare of Japan and a Grant for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (19770073).
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