The three complete mt genome sequences of C. hongkongensis
that we independently obtained were submitted to GenBank: accession No. EU672834
for oyster HN from Hainan, FJ593172 for oyster BH45 from Guangxi, and FJ593173 for oyster H50 from Fujian. The length of the complete mt genome of C. hongkongensis
reported by Yu et al
. is 16,475 bp. The length of all three mt sequences that we obtained is 18,622 bp, which is 2,147 bp longer than that of Yu et al
.'s. We aligned our sequences with that of Yu et al
. and other Crassostrea
species. We annotated our mt sequences according to that of C. gigas
with minor revisions [2
], and the results are presented in Table . Our sequence for C. hongkongensis
has exactly the same gene order and arrangements as C. gigas
, both containing the segment that is missing in Yu et al
.'s sequence. The segment contains four tRNA genes, a duplicated rrnS
and part of the split rrnL
. The split rrnL
is first discovered in C. virginica
and appears to be unique for oysters [2
Annotation of the mitochondrial genome of Crassostrea hongkongensis.
The three C. honghongensis
oysters used in our study were from diverse populations (Hainan, Guangxi and Fujian) covering the entire geographic range of this species as we know (Guo et al
., unpublished), and they were genetically identified using molecular markers prior to our study [3
]. We compared one of our sequences with Yu et al
.'s using BLAST http://blast.ncbi.nlm.nih.gov/bl2seq/wblast2.cgi
]. In the 16,475 shared nucleotides, there are 15 SNPs (single nucleotide polymorphisms) and the similarity between the two gnomes is 99.91%, suggesting that oysters used in our study and Yu et al
.'s study are all C. hongkongensis
. Sequence identity in major coding genes between our C. hongkongensis
sequences and that of C. gigas is shown in Table . Considerable differentiation has occurred between the two sister-species at some genes (i.e., gene identity of 75.1% for nad2) despite the identical gene order. Analysis of all four C. hongkongensis
mt sequences revealed 41 SNPs: 28 in coding and 13 in non-coding regions (Table ). Of the 19 SNPs from protein coding genes, only one is non-synonymous, suggesting strong purifying selection. The non-synonymous mutation occurred at the atp6
gene in Yu's sequence only, and further studies are needed to determine whether it is a true SNP or sequencing error.
Sequence identity of major coding genes between Crassostrea hongkongensis and C. gigas.
Single-nucleotide polymorphism (SNP) observed among four mitochondrial genome sequences of Crassostrea hongkongensis.
Yu et al. used ten pairs of primers to amplify the complete mt genome of C. hongkongensis (p. 11). We carefully studied the positions of each primer and located them in our mt genome sequences of C. hongkongensis (Figure ). It occurred to us that Yu et al. might have failed to amplify the gene block of K1-C-Q1-rrnL5'-N-rrnS2 because some of their primers were placed in a duplicated region. As shown in Figure , primer pair 1* is located in gene cob and rrnS1 (or rrnS2), primer pair 2* is completely located within the duplicated gene rrnS (rrnS1 or/and rrnS2), and primer pair 3* is located in rrnS2 (or rrnS1) and atp6 (primer pairs 1*, 2* and 3* correspond to the third, the fourth and the fifth primer pairs in Yu et al.' paper, Table ). Because these three primer pairs are either completely or partially (one of the two primers) located in the duplicated gene rrnS1 and rrnS2, they should theoretically amplify two fragments of different length, but in reality the smaller fragment may be preferentially amplified and sequenced. The length of shortest PCR products expected from the three primer pairs was 2,470 bp, 824 bp and 1,016 bp, respectively (Table ). Primer pair 2* was completely located in the duplicated gene rrnS (rrnS1 or rrnS2); thus they may directly concatenate the sequence between the duplicated gene and artificially lose the gene block of K1-C-Q1-rrnL5'-N-rrnS2 (Figure ). The block, 2,147 bp, may be too large to be amplified under competition with a smaller fragment.
Figure 1 Position map of the primers used in amplifying fragments of C. hongkongensis mitochondrial genome. Above the gene map are the three pairs of primers used by Yu et al. (2008) and below are the two pairs of primers designed to confirm the existence of the (more ...)
Primers used to amplify fragments of Crassostrea hongkongensis mitochondrial genome.
To test our hypothesis that the gene block between duplicated rrnS failed to amplify in Yu et al.'s study, we synthesized the three primer pairs used by Yu et al. (after removing mismatches based on our C. hongkongensis sequences to improve specificity). As expected, the three shorter products mentioned in Yu et al.' paper were successfully obtained (Table , Figure ). We increased the elongation time for PCR trying to obtain the longer fragments, but failed probably because of distance between the duplicated genes (2,147 bp) is too long. We designed two new pairs of primers targeting the block between the duplicated rrnS genes, with one primer of each pair located in the rrnL gene that was supposed to be absent according to Yu et al. (Table , Fig, ). The two new primer pairs designed by us successfully amplified and produced fragments of expected sizes, 2,658 and 1,905 bp (Table , Figure ), proving that the gene block between the duplicated rrnS genes are actually there. To further confirm that the two products both contain the duplicated rrnS, each product was used as PCR template for amplification with the primers 2* that amplifies rrnS only; both PCR produced a fragment of the expected size (824 bp), the same as using genomic DNA as template (Figure ). We also sequenced some of the fragments, and the sequences are the same as expected from the mt sequences we obtained. These results clearly demonstrate that the duplicated rrnS and the split rrnL exist in the mt genome of C. hongkongensis. There is no loss of the duplicated genes and the gene block between them. "Tandem duplication-random loss" is not a real feature of oyster mt genomes and has not occurred during the evolution of C. hongkongensis. The possibility of Yu et al. sequenced a rare mutant of C. hongkongensis is extremely low considering: 1) we sequenced three individuals from three diverse populations; 2) Yu and colleagues screened more than one individual; and 3) we duplicated their results with our samples. This is a clear case of PCR artifacts involving duplicated genes.
Figure 2 PCR products amplified with different primers and separated on agarose gel electrophoresis. P1 - P3 are the products amplified using the primer pairs 1* – 3* and P4, P5 are the products amplified with the primers 4, 5 with genomic DNA template; (more ...)