The complete plastid genome of the wild rice species Oryza brachyantha (Poaceae).

The whole plastid genome of wild rice (Oryza brachyantha) is characterized in this study. The genome is 134 604 bp in length and is arranged in a typical circular structure, including a pair of inverted repeats (IRs) of 20 832 bp in size separated by a large single-copy region (LSC) of 80 411 bp in length and a small single-copy region (SSC) of 12 529 bp in length. The overall GC content of the genome is 38.98%. One hundred and ten unique genes were annotated from the chloroplast genome, including 76 protein-coding genes, 4 ribosomal RNA genes and 30 tRNA genes. A total of 20 of these genes are duplicated in the IR regions, 13 genes contain 1 intron and 2 genes (rps12 and ycf3) have 2 introns.

The genus Oryza, is an ideal model system for plant comparative genomics, as well as studies of evolutionary and functional biology (Lu et al. 2009). Within Oryza, two species are cultivated and the remaining ∼22 wild species are wild. Ten distinct nuclear genome types, including six diploid (A, B, C, E, F and G) and four allotetraploid (BC, CD, HJ and HK), have been verified in this model genus (Ge et al. 1999). The cultivated rice has played an essential role in human nutrition and culture for millennia, and has important syntenic relationships with the other cereal species. As such, Oryza is among the most well-known and important study systems for Poaceae evolution and domestication (Lu et al. 2009). The wild relatives of Oryza have provided invaluable genomic resources for rice improvement and breeding (Dong et al. 2013). As the most distant wild relative of cultivated rice, O. brachyantha (F genome) is a basal lineage in Oryza, and may possess novel disease resistance and stress tolerance genes not found in more closely related species. Chen et al. (2013) found that O. brachyantha has the most compact genome within the genus Oryza, suggesting that a reduced genome is either plesiomorphic or genome reduction happened in this species since there is divergence from the most common recent ancestor of Oryza. Despite the numerous studies in Oryza, the complete plastid genome of O. brachyantha has not been assembled until now. By assembling the plastid genome of O. brachyantha, all plastid genome changes among Oryza species can now be examined. While plastomes are relatively small, they have supplied valuable informative markers in plant molecular systematics (Jansen et al. 2007; Wang et al. 2010; Wu & Ge 2012), biogeographical relationships among populations (Wang et al. 2011), plant DNA barcoding (Group CPBOL et al. 2011) and plant genetic transformation (Cui et al. 2011). In this study, we determined the whole plastid genome of O. brachyantha (Accession number from International Rice Research Institute as IRGC101232) by using next generation sequencing technology and Sanger sequencing.

By extracting the genome data (SRA046388) used for whole genome assembly from Chen et al. (2013), the trimmed and high-quality paired end (PE) reads were used to assemble the plastid genome from the CLC workbench (ver. 7.01 beta, CLC Inc., Rarhus, Denmark). We utilized reference mapping and de novo assembly methods to assemble the plastome by employing O. australiensis (NC_024608) as reference following the method from Wu and Tembrock (2015). We also employed the Sanger sequencing to verify the final assembly. This draft sequence was validated and manually corrected by mapping the raw PE reads, back to the complete chloroplast genome of O. brachyantha (KT992850). Structural features and genes in the chloroplast genome were predicted following the method from Wu and Ge (2016).

The 134 604 bp long O. brachyantha plastome possesses a typical plastid double-stranded circular arrangement with a quadripartite structure common to most of the land plant plastomes (Raubeson & Jansen 2005; Wu & Ge 2012; Wu & Ge 2016). The large single copy (LSC) regions are 80 411 bp in length and the small single copy (SSC) regions are 12 529 bp in length separated by two inverted repeats (IRs) of 20 832 bp in size. The O. brachyantha plastome was found to contain 128 functional genes: of which 110 are in single copy and 18 are duplicated in the IR regions. Among the 110 annotated single-copy genes, 76 are protein-coding genes, 30 tRNA genes, and four rRNA genes, amounting to 44.19%, 2.13%, and 6.83% of the genome, respectively. The remaining 46.85% of the genome is made up of non-coding introns and intergenic spacers. Similar to other Poaceae plastomes (Wu & Ge 2012; Wu & Ge 2016), the O. brachyantha plastome is also AT-rich (61.02%). The four rRNA genes are all located in the IR regions. Twenty one tRNA genes are located in the single-copy region, whereas the other nine are located in the IR. Fifteen genes contain introns: ycf3 contains two introns and the rest of the genes contain a single intron. Rps12 is trans-spliced, with one of its exons in the LSC region (5′ end) and the other two in the IR regions (3′ end) separated by an intron. NdhH spans the IR and SSC junction. The matK gene was located within the intron of trnK-UUU. In the O. brachyantha plastome, the pairs of genes atpB-atpE, psbC-psbD and ndhC-ndhK had 4 bp, 53 bp and 10 bp overlapping regions, respectively.

Phylogenetic analysis, using the whole plastome alignment from 13 published Oryza species including O. brachyantha and two outgroup Oryzeae species, was performed using neighbour-joining (NJ) analysis in MEGA 6.0 (Tamura et al. 2013). Our phylogenetic analysis of plastomes divided the 13 Oryza species into the four predetermined genome groups (Figure 1). As expected, O. brachyantha was resolved as an early diverging lineage in Oryza. The phylogenetic relationships of the Oryza plastomes were identical to previous phylogenetic studies using nuclear genetic markers (Ge et al. 1999; Tang et al. 2015).

Figure 1.

NJ phylogenetic tree of O. brachyantha with other 14 species in the Oryzeae order based on complete plastome sequences. Numbers in the nodes are bootstrap values from 1000 replicates. The plastome of Rhynchoryza subulata (NC_016718), a species from Zizaniinae subtribe, was set as an outgroup.