Wang et al. BMC Genomics 2011, 12:289 http://www.biomedcentral.com/1471-2164/12/289 appeared earlier and occurred to a more significant degree (Figure 1D). We noticed that the seedlings exposed to 46°C for 1 h were etiolated on the 12th day after the heat treatment (Figure 1C), while those exposed to 47°C for 1 h etiolated on the 5th day after heat treatment, revealing that 47°C for 1 h is a critical temperature and time point for this genotype. Consider- ing the differences in the early gene expression events and physiological reactions observed in the plants, we chose the heat treatment of 46°C (HT) and the moder- ate temperature (MT) for the small RNA deep sequen- cing experiments. We expected that the temperature of this heat treatment was high enough to affect the bio- genesis of small RNAs but did not cause significant morphological and physiological changes to the seed- lings. The heat-treatment method is simple, easy to manage, accurate and only required a short length of time. Using this method, we have selected more than 20 lines of Chinese cabbage with heat resistance. Small RNAs generated from the chloroplast genomes of Chinese cabbage For a global view of small RNAs expressed in Chinese cabbage, we chose to collect material from the above ground portions of HT and MT seedlings and sequenced RNA fractions of 9 to 36 nucleotides (nt), using Illumina Genome Analyzer and mirVana™ miRNA Isolation Kit (Ambion, Inc). Two replicates of HT treatment generated 14.6 million and 12.8 million small RNA reads, while those of the MT treatment pro- duced 14.7 and 11.3 million small RNA reads, indicating high quality achieved. For a convenient comparison, the data from HT with 14.6 million and that from MT with 14.7 million reads were used for the analysis. To select chloroplast small RNAs from the whole population of reads, the trimmed sequences were com- pared to a complete chloroplast genome and a nuclear genome of Chinese cabbage by BLAST after the removal of the adapters. The small RNAs that perfectly-matched the chloroplast genome were retained. In total, we obtained 2,162,001 and 1,278,752 csRNAs from MT and HT, respectively, which represents 116,210 and 78,299 unique small RNA sequences, and account for 15% and 10% of the total small RNA population, respectively (Fig- ure 2A and Table 1). Since the chloroplast genome sequence of Chinese cabbage had not been annotated, we matched all small RNA sequences with the Arabidopsis chloroplast genome for further genomic origin analyse. The two species share 98% identity in the DNA sequences of their chloroplast genomes. In this case, more than 95% of the small RNAs from Chinese cabbage are matched perfectly with the Arabidopsis chloroplast genome (Table 1). The small RNAs that are perfectly Page 3 of 14 Figure 2 Proportion of csRNAs in each sequence dataset. (A) Percentages of Chinese cabbage csRNAs. (B) Percentages of Arabidopsis csRNAs. (C) Venn diagram of Chinese cabbage csRNAs and Arabidopsis csRNA datasets. The numbers represent the number of unique csRNAs in each plot. matched with both genomes of two species were desig- nated as chloroplast-related small RNAs (csRNAs). Since some fractions of chloroplast DNA sequences match the nuclear or mitochondrial genome, the csRNAs should include those from the nucleus and mitochondria. To estimate the proportion of chloro- plast-specific small RNAs that originated from chloro- plast genome rather than nuclear and mitochondrial genomes, we established the chloroplast-related small RNA dataset of Arabidopsis thaliana (Landsberg erecta ecotype) paralleled to that of Chinese cabbage by small RNA deep-sequencing, and then calculated the abun- dance of the csRNA sequences in the Arabidopsis chlor- oplast genome. In total, 787,272 csRNA sequences from Arabidopsis perfectly matched its chloroplast genome