Wang et al. BMC Genomics 2011, 12:289 http://www.biomedcentral.com/1471-2164/12/289 Page 4 of 14 Table 1 Chinese cabbage Small RNA reads originated from chloroplast genome Dataset HT MT Origin Total Total Mapped to the Chinese cabbage chloroplast genome Mapped to both Chinese cabbage and Arabidopsis chloroplast genomes a Mapped to the Chinese cabbage chloroplast genome Mapped to both Chinese cabbage and Arabidopsis chloroplast genomes Unique sequence (Reads) Abundance (Reads) 4,439,626 78,299 59,261 3,816,252 116,210 89,878 14,622,224 1,278,752 1,225,538 14,690,798 2,162,001 2,095,078 a: Chinese cabbage csRNA dataset (Additional File 1). By removing those sequences that matched the nuclear and/or mitochondrial genomes, we obtained the chloroplast-specific small RNAs of Arabi- dopsis, which account for 86% of the unique csRNAs. The size distribution and the origin composition of csRNAs were consistent with those of chloroplast-speci- fic small RNAs (Additional File 2). A high proportion of chloroplast-specific small RNAs and the similar compo- sitions revealed that the csRNA dataset is representative of its chloroplast-specific small RNA dataset. Consider- ing the close relation between two species, the property of csRNAs in the Chinese cabbage dataset should be representative of the entire population of chloroplast- derived small RNAs. The distribution of csRNAs over the genome csRNAs of Chinese cabbage exhibited wide variations in length, from 9 to 36 nt (Figure 3A and 3B). Among the small RNAs mapped to the chloroplast genome, the most numerous unique csRNAs were 24 nt in length (Figure 3A), but 22 nt small RNAs were the most abun- dant in MT seedlings (Figure 3B). csRNAs were classi- fied into four types: mRNA, rRNA, tRNA, and intergenic RNAs (igRNA), according to the types of RNA from which csRNAs are derived. Among them, the csRNAs derived from rRNA are the most abundant, and those from mRNA are the least prevalent (Figure 3C and Additional File 3). Further analysis showed that a large proportion of rRNA-derived csRNAs were 18 to 25 nt in length with three peaks of 18-19, 21-22 and 25 nt, while the csRNAs derived from tRNA showed a maximum of 29 nt with 2 smaller peaks at 23-24 and 32 nt (Additional File 4). In addition, mRNA-derived csRNAs are generally 21-24 nt in length (Additional File 4E and 4F), similar to that reported by the early work on nuclear-expressed genes. The csRNAs originating from igRNAs were on average 22 nt in length, which roughly match the numbers of unique csRNAs (Addi- tional File 4H and 4G). We noticed that some csRNA families were very large as they shared the same core sequence, typically 9-15 nt in length, with 5’ or 3’ exten- sions of variable lengths (Additional File 5). This feature did not fit with our canonical knowledge of small RNA biogenesis, as the precursor that would be loaded into the Dicer-like complex seems variable in size. csRNAs from 3’ ends of rRNA and 5’ ends of tRNA The chloroplast genome contains two inverse repeats, from where all fragments of rRNA are transcribed [25]. Each repeat consists of four types of rRNA: 23S, 16S, 5S and 4.5S. To examine the distribution of csRNAs along rRNA sequences, we divided the transcribed regions of rRNA into 5% blocks from the 5’ to 3’ ends, and plotted the positions of csRNA ends into them. Interestingly, an enrichment of the rRNA-derived-csRNAs were fallen into the last 5% block of chloroplast rRNA (Figure 4A), even showing a remarkable peak at the 3’ ends of rRNA (Figure 4C). csRNAs from 4.5S rRNA sequences account for a substantial portion of csRNAs originating from rRNA (~70%). An enrichment of the csRNAs from 4.5S and 5S rRNA were fallen into the last 5% block of 4.5S rRNA and 5S rRNA, while those from 16S and 23S rRNA were not (Additional File 6A-D). Moreover, there was a remarkable abundance peak for the last nucleotide of the mature 4.5S rRNA and one outer nucleotide of the mature 5S rRNA 3’ terminus, respectively (Addi- tional File 6EE-H), indicating that the biogenesis of csRNAs in the 4.5S and 5S rRNA molecules are 3’ end- preferential. There are 20 types of tRNAs in the chloroplast, repre- senting the entire amino acid (anti) codons [25]. Simi- larly, we divided the nucleotide sequences of the tRNAs into 5% blocks from the 5’ to 3’ termini, and plotted the positions of csRNA ends. Interestingly, the csRNAs derived from most of the chloroplast tRNA sequences (except tRNA-Ser, tRNA-Thr and tRNA-Trp) exhibited an abundant peak in the first 5% block, which accounted for more than 95% of the tRNA-derived csRNAs (Figure 4B). Moreover, the first nucleotide of these csRNAs were predominantly located at the first nucleotide of the mature chloroplast tRNA sequences (Figure 4D), reveal- ing that the biogenesis of csRNA in tRNA molecules is specific to the 5’ end. These results are consistent with the previous reports, in which the majority of small RNAs originated from the rDNA locus [26] and the 3’ ends of rRNA [27], and