Oryza rufipogon specific genes
Several lines of evidence suggest that Oryza sativa was derived from O. rufipogon (e.g., Yang et al. 2011, Yang et al. 2012). Therefore, it is of interest to find genes that contributed to make differences between O. sativa and O. rufipogon. In particular, detection of genes lost during the domestication process will help us understand the adaptation and diversification of wild and cultivated rice. Thanks to the wealth of genome and transcriptome sequences of rice, we are capable of identifying genes lost in rice cultivars through genome-wide comparative sequence analysis.
Among the 2,044 full-lenght cDNA (FLcDNA) sequences of O. rufipogon determined by Lu et al. (2008), 87 could not be mapped to either japonica (Nipponbare) or indica (93-11) genome. We conducted PCR analysis for the 87 sequences and found that at least the following 15 genes are missing in the Nipponbare genome.
Of these genes, five (CT842002, CU405880, CU406257, CU406702, and CU861681) were up/down-regulated in wounding and/or submergence conditions (See the following figure).
Figure.O. rufipogon-specific FLcDNAs associated with stress (wounding and submergence) conditions. J: O. sativa (japonica); Or: O. rufipogon; C: control; W: wound treatment; S: submergence treatment.
MATERIALS AND METHODS
Seeds of O. rufipogon W1943 were treated at 50-54 °C for five days to break dormancy. The treated seeds were dehulled, sterilized, and grown for three weeks. Seeds of Nipponbare were grown for one week. Leaves of one-week-old Nipponbare and three-week-old W1943 seedlings were sliced for the extraction of genomic DNA. To extract genomic DNA, the sliced leaves were treated by liquid nitrogen, powdered, added PCR buffer (200 mM Tris-HCl (pH 8.0), 250 mM NaCl, 25mM EDTA, 0.5% SDS), and incubated for one hour at 65 °C. The solution was added chloroform, shaken for 20 minutes, and centrifuged for five minutes. After centrifugation, the aqueous phase was transferred to a new tube and DNA was precipitated by isopropanol. Precipitated DNA was dried and dissolved in TE solution.
Selection of sequences
The 2,044 O. rufipogon FLcDNA sequences were isolated and sequenced by Lu et al. (2008) and their data were downloaded (accession #: CT841557-CT841684; CT841686-CT841707; CT841710-CT841954; CT841956-CT842008; CU405560-CU405627; CU405629-CU405654; CU405656-CU405706; CU405708-CU405710; CU405712-CU405714; CU405716-CU405717; CU405719-CU405720; CU405722-CU405729; CU405731-CU405880; CU405882-CU405928; CU405930-CU406064; CU406066-CU406249; CU406251-CU406335; CU406337-CU406954 and CU861673-CU861883). Repeats in O. rufipogon FLcDNAs were masked by RepeatMasker with the library available in MIPS Repeat Element Database. PolyA tail and vector sequences were also masked using customized Perl scripts. After masking, FLcDNAs were mapped to the japonica (cv. Nipponbare) and indica (cv. 93-11) genomes by the criteria that identity >80% and coverage >80% using BLAST version 2.2.16. Those that could not be mapped to either japonica or indica genome were candidates for further PCR analysis. Primer sets were designed based on those unmapped sequences using Primer3 (Rozen and Skaletsky 2000).
PCR analyses and sequencing
DNA fragments were amplified by PCR using GoTaq® Green Master Mix
(Promega, Madison, WI USA) or PrimeSTAR GXL DNA polymerase (Takara,
Otsu, Japan). The PCR products were used for gel electrophoresis to
check whether the amplification was successful; DNA fragments that were
amplified from O. rufipogon but could not be amplified from Nipponbare
were further extracted using QIAquick Gel Extraction Kit (Qiagen,
Valencia, CA USA) and purified by QIAquick PCR Purification Kit (Qiagen,
Valencia, CA USA) to determine the sequences. Sequencing was performed
following the protocol of BigDye® Terminator v3.1 Cycle Sequencing Kit
(Applied Biosystems, Foster City, CA USA).
The SuperScript™ One-Step RT-PCR with Platinum® Taq System (Invitrogen, Carlsbad, CA USA) was used for amplification of O. rufipogon-specific genes by RT-PCR.