The genome sequence data for the CC-genome species, the chromosome-level assembly sequence of O. officinalis (Shenton et al., 2020), was obtained from the DNA Databank of Japan (DDBJ: https://www.ddbj.nig.ac.jp/index-e.html). The genome sequences of four rice cultivars (IR8, IR64, Minghui 63, and Zhenshan 97) were downloaded from the NCBI nucleotide database. Inclusion of several cultivar sequences in the multiple sequence alignments was intended to select common InDel regions among cultivars rather than variety specific InDels. So that the potential polymorphic markers can show high polymorphism with many other rice cultivars. Approximately 20-kb bait sequences of Nipponbare were prepared from the RAP-DB (https://rapdb.dna.affrc.go.jp). To avoid long repeat regions such as transposable elements in bait sequence, the bait sequences were manually selected by using display option of ‘Repeat region’ under JBrowse, RAP-DB. The next bait sequences were manually moved with ~2Mb intervals along each chromosome, ensuring coverage across the genome. Orthologous regions were isolated from the bait sequence using the BLAST tool under NCBI Genome Workbench Software (Default parameters with Word Sizes: 70-120). Subsequently, multiple sequence alignment was performed using the web-based tool mVISTA (http://genome.lbl.gov/vista/), and polymorphic regions observed in the aligned sequences were highlighted and exported into a text file format using the BioEdit software (Hall, 1999).

The multiple sequence alignment exported from the BioEdit software was opened using Microsoft Word. InDel regions containing gaps greater than 20 base pairs were manually screened among O. officinalis and the cultivars for clear separation in ~2% agarose gel. Forward and reverse primers were manually designed on the conserved regions flanking the polymorphic InDel sites to ensure proper primer annealing across all the species analyzed. Primer design followed a specific criterion: (1) GC content of approximately 50%; (2) annealing temperature around ~55°C; and (3) expected product sizes ranged from 100 to 500 bp. These parameters were laid out to standardize PCR conditions across all markers. Primer sequences were checked for redundancy using BLAST in RAP-DB, and only primers with unique hits were selected.

To validate the newly developed marker sets, PCR followed by agarose gel analyses was conducted using genomic DNA from wild and cultivated rice accessions. This panel included four accessions each of the CC-genome species, O. eichingeri, O. officinalis, O. rhizomatis, two accessions of BBCC-genome species O. minuta, and two Asian cultivated varieties: O. sativa spp. indica cv. IR24 and O. sativa spp. japonica cv, Nipponbare). All plant materials for the marker validation are listed in Table 1. Seeds of the wild rice species were obtained from the IRRI Genebank (https://gringlobal.irri.org/gringlobal/search), and wide-hybridization materials with CC-genome containing species were previously developed and maintained by the GIV laboratory at the IRRI. All the plant materials were grown in the glasshouse for wild rice species at the IRRI headquarters, Philippines.

Leaves approximately 2–4 cm were collected from the plant materials. Each leaf was directly placed in a 2-mL microcentrifuge tube containing two stainless steel balls inside. DNA extraction was carried out using a modified simple protocol based on Kim et al. (2016). A volume of 400 μL of TPE buffer (100 mM Tris-HCl, pH 9.5, 1 M KCl, 10 mM EDTA, pH 8.0) was added to each tube. Samples were then homogenized using a 2010 Geno/Grinder^® at 1000 RPM for 7 min. Following homogenization, the samples were incubated in a hot water bath at 65°C for 60 min. After incubation, tubes were centrifuged at 13000 × g for 15 min. The supernatant, containing the extracted genomic DNA of the samples, was transferred to 1.5-mL microcentrifuge tubes and stored at 4°C. For PCR analysis, each DNA sample was diluted by transferring 30 μL of crude DNA extracts into a 96-well plate containing 120 μL of double-distilled water and used as template DNA.

For PCR analysis, a 15 μL PCR mixture was prepared containing the following components: 10x PCR buffer, 200 μM of deoxyribonucleotide triphosphate (dNTP), 0.50 μM each of the forward and reverse primers, 1 unit of Taq DNA polymerase (BioFact, https://www.bio-ft.com/eng/index.html), 2 μL of the DNA and double distilled water to reach the final volume. Approximately 8 μL mineral oil was added to the reaction to avoid evaporation during thermal cycling. PCR was performed using thermal cyclers (Applied Biosystems VeritiTM 96- or 384-Well Fast Thermal Cycler, http://www.thermofisher.com) with reactions placed in either 96- or 384-well plates. The thermal cycling profile used for marker validation was as follows: an initial denaturation at 94°C for 2.5 min; 35 cycles of 95°C for 25 s, 55°C for 25 s, 72°C for 50 s; followed by a final extension at 72°C for 5 min. PCR products were separated on ~2% agarose gels.

The physical positions of the developed markers were visualized on the 12 rice chromosomes using the web-based tool PhenoGram (http://visualization.ritchielab.psu.edu).

We used a previously established strategy of multiple sequence alignment and primer design (Hechanova et al., 2021; Malabanan-Bauan et al., 2023) to develop evenly distributed genome-wide InDel markers that can distinguish alleles between cultivated rice (O. sativa) and CC-genome wild rice species. Specifically, bait sequences were obtained from the rice reference genome available at RAP-DB and used to retrieve orthologous genomic regions from the publicly available CC-genome species (O. officinalis) and cultivated rice varieties (AA-genome).

In certain instances, orthologous regions were not detected. This indicates possible absence or high divergence of those regions in wild rice species or some cultivars. Only orthologous sequences shared between O. officinalis and the two cultivars were retained for multiple sequence alignment. Alignments were generated using the mVISTA web-based tool, and sequence variations such as SNPs and InDels were identified using BioEdit. For clear separation in agarose gel, InDel regions showing at least a 20-bp gap between O. officinalis and the cultivars were selected for marker development.

Marker development was initiated from the tip of the short arm of each chromosome and was subsequently moved down in ~2Mb intervals to the next markers for uniform genomic coverage. A total of 182 markers were designed and distributed across the 12 rice chromosomes. Expected PCR amplicon sizes for each marker were calculated based on the alignment results for the two rice cultivars and O. officinalis (Supplementary Table S1).

Each marker was named using the format CxxPxxxxx, where “Cxx” denotes the genome type and chromosome number, and “Pxxxxx” represents the physical location (in kilobases) on the chromosome of the rice reference genome, Nipponbare (Figure 1A). This naming convention facilitates easy identification of both chromosomal and physical locations of each marker.

To validate the developed markers, all 182 markers underwent PCR amplification followed by agarose gel analysis using genomic DNA from a japonica cultivar (Nipponbare), indica cultivar (IR24), four accessions each of CC-genome wild rice species O. eichingeri, O. officinalis, O. rhizomatis, and two accessions of BBCC-genome wild rice species O. minuta. Out of the 182 markers, 172 markers (94.5%) successfully amplified and showed polymorphism between rice and one accession of CC-genome germplasm, with an estimated average marker interval of ~2.17Mb across the rice reference genome (IRGSP-1.0), while 10 markers showed no/weak amplification or produced unexpected multiple bands. All the gel images of the 182 markers and marker information are shown in the Supplementary Figure S1 and Supplementary Table S1, respectively.

Among the 12 accessions under CC-genome, three O. officinalis accessions (Offi_C06, Offi_C07, Offi_C08) and one O. rhizomatis accession exhibited the highest number of polymorphic markers (172) when compared against Nipponbare (Table 1). Similarly, two O. officinalis accessions (Offi_C07, Offi_C08) displayed the highest number of polymorphic markers (171) against IR24. In contrast, lowest number of polymorphic markers for CC-genome were observed in two O. eichingeri accessions (Eichi_C02, Eichi_C04) and one O. rhizomatis accession (Rhizo_C11) with 164 and 162 polymoprhic markers when compared to Nipponbare and IR24, respectively. The CC-genome markers were also tested with two accessions of the BBCC-genome species, O. minuta. The results showed high polymorphisms (153 to 159 polymorphic markers) between cultivars and O. minuta (Table 1). The 172 markers exhibiting polymorphisms were evenly distributed across all 12 chromosomes (Figure 1B). In addition, 87 previously reported polymorphic markers developed and validated by Hechanova et al. (2018) were mapped alongside the newly designed polymorphic markers (Figure 1C), providing a comprehensive marker resource with an estimated average marker interval of ~1.44Mb for genetic studies in CC- and BBCC-genome wild rice species.

By using the marker validation data, we isolated the species-specific markers that can discriminate the three CC-genome species. Two markers (C04P15867 and C07P15240) showed specific band patterns for O. eichingeri accessions, and three markers (C10P16655, C07P27830, and C08P02385) were specific for O. officinalis accessions (Figure 2). However, we were not able to find O. rhizomatis specific markers. These five markers would be useful to determine the species within the CC-genome species.

We tested the newly developed CC-genome markers with the CC-genome-containing introgression lines in rice. Six markers showing polymorphism between rice and CC-genome species were randomly selected and applied for the detection of O. rhizomatis introgressions from 14 individual BC₁F₁ plants derived from the elite rice breeding line (IR31917-45-3-2) x O. rhizomatis cross. As expected, all six markers could detect introgressions of different chromosomes (Figure 3A). In addition, the same six markers were used to check true F₁ hybridity from the cross between IR64 and O. latifolia (CCDD-genome) which contains CC genomes. Interestingly, all six markers also showed polymorphism between IR64 and CCDD-genome species (O. latifolia) and the F₁ plants possessed both parental bands (Figure 3B), suggesting all three F₁ plants were true hybrids. These results suggested that the newly developed CC-genome markers might be useful to harness the CC-genome species and also the CC-genome containing species such as BBCC and CCDD-genome species.