2.1 Plant Materials
Six distinct groups within the domesticated-weed-wild complex (DWWC) in the genus Oryza , sharing a common ancestry, were sampled from Sri Lanka for this study. These groups include inbred rice, landraces, feral rice, weedy rice, O. rufipogon , and O. nivara .
Seeds of 42 inbred rice varieties (206 individuals; Table S3) were collected from the Rice Research and Development Institute (RRDI) of Sri Lanka. Seeds of 31 landraces (151 individuals) with 4-5 individuals per variety were collected from the seed bank of the Plant Genetic Resources Centre (PGRC) at Gannoruwa, Peradeniya, Sri Lanka (Table S2). Both inbred and landraces (5-10 seeds of each cultivar) were planted in equal-sized pots (9 cm diameter top, 6.3 cm diameter base, 8.7 cm depth, and capacity: 0.37 liter) filled with sieved paddy soil and grown under greenhouse conditions without added fertilizer. After two weeks, the young tender leaves (3-5 leaves) from randomly selected 3-5 plants were harvested from each cultivar and stored at -80 °C until extraction of DNA.
The other free-living Oryza populations, including weedy, feral, and wild rice, were sampled from various locations across Sri Lanka. To prevent repeated samples of the same genets (clones), the distance between sampled individuals was maintained at least 10 m apart. The homogeneity of inbred rice makes it easy to distinguish weedy rice by its characteristic traits, such as differences in seed and panicle features, typically exhibiting taller stature, profuse tillering, a more open or spreading growth habit, and weaker culms. Weedy rice plants were collected from 20 major rice-growing areas (20 populations) in Sri Lanka that had a recent history of heavy weed infestation, as confirmed through personal communication with the Department of Agriculture, Sri Lanka (see Fig. 1). One population was defined as approximately 25 weedy rice plants randomly sampled from each field, which covered an area of approximately one hectare. Thus, a total of 500 individual weedy rice plants were collected across the entire geographical distribution in Sri Lanka. The sampled rice fields infested with weedy rice were at least 30 km apart from each other. For this study, six O. nivara (183 individuals) and five O. rufipogon (132 individuals) populations were sampled, comprising 18 to 31 individuals per population and representing the diverse wet, dry, and intermediate zones of Sri Lanka. Feral rice consisted of O. sativa populations that emerge spontaneously in paddy fields abandoned for approximately 10 years. These plants are presumed descendants of previously cultivated inbred or landrace varieties. Unlike weedy rice, feral rice is not considered a weed in paddy fields. In this study, we sampled seven feral rice populations, collecting 24 individuals per population (168 individuals in total), from abandoned paddy fields for further analysis. Green leaf samples were collected from wild, weedy and feral rice in theMaha season from late February to March 2016. The green leaves were collected individually in the field, placed in zip-lock bags containing silica gel, and stored at 0-4 °C until DNA extraction. Information on population codes, the number of individuals sampled, locations, and GPS readings of the weedy rice populations are given in Table S1.
Fig. 1. Geographical locations of 20 weedy rice (blue circles), five Oryza rufipogon (green triangles), six Oryza nivara(yellow triangles) and seven feral rice (the red squares) populations sampled in this study. Dark lines separate the climatic zones in this study. Detailed information on these populations is provided in Table S1.